Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Up-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Down-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Reporter assays detected stimulation of p53-mediatd transactivation by MEG3 deletion mutants M1, M1+M2, M3, M2+M3 in HepG2 cells. The value are means of three independent experiments ±S.D, * P<0.05. (B) Cropped blots show the increased level of p53 protein 48h after transfection of the pcDNA-MEG3 in HepG2 cells. (C) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in HepG2 cells (0, 0.5,1, 2h) overexpressing MEG3. (D) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in doxo-induced HepG2 cells (0, 2,4, 6h)overexpressing MEG3.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Transfection, Over Expression

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 incubated with HEK293 cell extract transfected with Flag-p53 vector. (B) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 and deletion mutations RNA incubated with doxo-induced HepG2 cell extract (MEG3 deletion mutants M1, M1+M2, M3, M2+M3 are shown in ). (C) In vitro transcribed biotinylated MEG3 retrieved purified GST-p53 but not GST. (D) RIP experiments were performed using an antibody against the p53 on extracts from doxo-induced HepG2 cells. The purified RNA was used for qRT-PCR, and the enrichment of the lncRNA MEG3 was normalized to GAPDH (upper, western blot of p53 protein after immunoprecipitation). Data was relative to mock-IP (IgG). (E) A series of p53 deletion mutants which were flag-tagged was treated as in (A), and association was detected by anti-Flag. Up represents successful expression of p53 deletion mutants. Down represents in vitro transcribed biotinylated MEG3 RNA was incubated with HEK293 cell extract which was transfected with p53 deletion mutants and associated proteins were detected by anti-Flag.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Western Blot, In Vitro, Incubation, Transfection, Plasmid Preparation, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) SK-Hep–1 hepatocellular carcinoma cell lines with stably expression of MEG3 were determined by qRT-PCR. The value are means of three independent experiments ±SD, *** P<0.001. (B) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH. The bars represent the relative fold change of these genes in SK-Hep–1 after transfection with pcDNA3.0-MEG3 compared with blank vector pcDNA3.0. (C) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH after knockdown p53 level in HepG2 cells. The bars represent the relative fold change of these genes in HepG2 cells after co-transfection of pcDNA3.0 and NC, pcDNA3.0-MEG3 and NC, pcDNA3.0-MEG3 and sip53, pcDNA3.0 and sip53, respectively. (D) A schematic diagram illustrating how MEG3 can function as tumor suppressor through interactions with p53.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Cotransfection

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: (A) Expression level of CD132, CD133, OCT3/4, NANOG, and EpCAM in RCC-41-PDX-1/CD132 + sorted cells. Results are representative of those obtained in three experiments. (B) Expression level of CD133, OCT-3/4, NANOG, and EpCAM in RCC-41-PDX-2/CD133 + and RCC-41-PDX-2/CD133 − sorted cells. Results are representative of those obtained in three experiments. For (A) and (B) , grey histograms correspond to cells incubated with the isotype-matched control antibody, and black outline histograms correspond to the analyzed markers. Grey numbers within panels correspond to Mean Fluorescence Intensity (MFI) of cells incubated with the isotype-matched control antibody, while black numbers correspond to MFI of the analyzed markers.

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Expressing, Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: (A) The aldehyde dehydrogenase (ALDH) activity in sorted RCC-41-PDX-1/CD132 + , unsorted RCC-41-PDX2, RCC-41-PDX-2/CD133 + and RCC-41-PDX-2/CD133 − was measured by flow cytometry (10 000 events). ALDH activity was measured in the absence and presence of the specific ALDH inhibitor DEAB following manufacturer's instructions. One experiment representative of three is shown. (B, C) Spheroïd formation following limiting dilution assay of the indicated cells. Data represent mean values of five replicates for two experiments; error bars correspond to 95% confidence intervals. (D) Clonogenic activity at limiting dilution: sorted RCC-41-PDX-1/CD132 + , RCC-41-PDX-2/CD133 + and RCC-41-PDX-2/CD133 − were plated in the absence of serum at a density of 1 to 10 cells per well in 24-well plates containing DMEM-LG medium. After 2 weeks, each well was examined under a light microscope, and the total number of wells with colonies was determined. Data represent mean values of three replicates for three experiments; error bars correspond to 95% confidence intervals.

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Activity Assay, Flow Cytometry, Limiting Dilution Assay, Light Microscopy

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: (A) Microarray analysis of CD133 + and CD133 − RCC41-PDX-2 cells. Two independent biological replicates are represented. (B, C) Comparative mRNA expression of ERBB receptors (B) and tumor suppressor genes (C) in CD133 − and CD133 + RCC41-PDX-2 cells by RT-qPCR. Presented results are mean of three independent experiments with * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Microarray, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: (A) Flow cytometry analysis of CD133 expression in RCC-41-PDX-1/CD132 + cell suspensions recovered from serial xenografts in SCID mice. Red outline histograms correspond to analyzed markers, and blue outline histograms correspond to cells incubated with the isotype-matched control antibody. (B) Vessel staining (CD31) and serial tumor formation by RCC-41-PDX-1/CD132 + cells subcutaneously injected in SCID mice. Top row : Representative hematoxylin and eosin staining showing the morphological appearance of an undifferentiated renal carcinoma with rare clear cells. Middle row : Representative immunohistochemistry of serial tumor sections showing positivity for human CD31 using a mAb that did not cross-react with the mouse CD31 (tumor vessels) and human CD133. CD133 + cells show a typical peri-vascular distribution. Bottom row : Representative immunohistochemistry of serial tumor sections showing positivity for human EpCAM. Original view × 40 (top) and × 60 (middle and bottom). Data are representative of 3 experiments with similar results. (C) Quantitative PCR analysis of E-cadherin ( CDH1 ) and Vimentin ( VIM ), expression in RCC-41-PDX-1/CD132 + cells. Data were normalized using TATA binding gene mRNA (TBP), as endogenous control. (D) Flow cytometry analysis of CD133 and EpCAM expression in RCC-41/PDX-1/CD132 + -1.1 cell suspension recovered from serial xenografts in SCID mice.

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Flow Cytometry, Expressing, Incubation, Control, Staining, Injection, Immunohistochemistry, Real-time Polymerase Chain Reaction, Binding Assay, Suspension

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: Tumor formation of unsorted RCC-41-PDX-2, and sorted RCC-41-PDX-2/CD133 + and RCC-41-PDX-2/CD133 − cells in SCID mice as a function of number of injected cells ( n = 4 per group)

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Injection

Journal: Oncotarget

Article Title: Isolation and characterization of renal cancer stem cells from patient-derived xenografts

doi: 10.18632/oncotarget.6266

Figure Lengend Snippet: (A) Tumor formation of unsorted RCC-41-PDX-2, and freshly sorted RCC-41-PDX-2/CD133 − and RCC-41-PDX-2/CD133 + cells in SCID mice as a function of number of injected cells 10 2 and 10 3 cells of each cell subtype were subcutaneously injected into SCID mice ( n = 4 per group). Represented is the time for evaluation of tumor volume (mm 3 ). Data are representative of 3 independent experiments with similar results. (B) Representation of tumor growth rate using survival curve. Death corresponds to a xenografted tumor reaching 1500 mm 3 . Data are representative of 3 independent experiments with similar results. *and **in (C) indicates respectively p < 0.05 and p < 0.005 by the log-rank test.

Article Snippet: All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA).

Techniques: Injection

Journal: Arthritis Research & Therapy

Article Title: Elevation of α-1,3 fucosylation promotes the binding ability of TNFR1 to TNF-α and contributes to osteoarthritic cartilage destruction and apoptosis

doi: 10.1186/s13075-022-02776-z

Figure Lengend Snippet: The α-1,3 fucosylation of TNFR1 mediated by FUT10 impacted the binding capacity to TNF-α. a Chondrocytes were transfected with siRNA and treated with or without TNF-α for 48 h, and the protein level of TNFR1 was determined by Western blotting. β-Tubulin was used as an internal control. b The relative expression level of TNFR1 from three experimental replications was normalized to β-tubulin and compared based on one-way ANOVA. c Immunoblot and lectin blot reactivity of TNFR1 immunoprecipitated from chondrocytes treated as described above. d The fucosylation of TNFR1 was normalized to the expression of TNFR1 and compared using one-way ANOVA. e Immunoblot and lectin blot reactivity of TNFR1 immunoprecipitated from cartilage of OA patients and normal controls. f The fucosylation of TNFR1 was normalized to the expression of TNFR1 and compared based on a paired t test . g A schematic diagram of the fabrication of the TNFR1 antibody microarray. h TNFR1 was immunoprecipitated from chondrocytes transfected with siRNA (low level of α-1,3 fucosylation) or induced with TNF-α (high level of α-1,3 fucosylation), and the scanned images were obtained for the analysis of the binding ability of TNFR1 with different levels of fucosylation to TNF-α. The spots of the TNFR1 antibody are marked with white boxes. i The fluorescence intensities of spots were extracted by Genepix 7, and the binding ability of TNFR1 was compared based on fold change and t test . The data are presented as the mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: The primary antibodies used in this study were as follows: (i) rabbit polyclonal anti-FUT10 (Proteintech, Wuhan, China), (ii) rabbit polyclonal anti-MMP-13 (Proteintech), (iii) rabbit polyclonal anti-COL2A1 (Proteintech), (iv) mouse monoclonal anti-IL-1β (Proteintech), (v) mouse monoclonal anti-NF-κB p65 (Proteintech), (vi) mouse monoclonal anti-phospho-NF-κB p65 (Ser536, CST), (vii) rabbit polyclonal anti-IKBA (Proteintech), (viii) rabbit polyclonal anti-phospho-IKBA (Ser32, CST), (ix) mouse monoclonal anti-P38 MAPK (Proteintech), (x) rabbit monoclonal anti-Phospho-p38 MAPK (Thr180/Tyr182, CST), (xi) mouse monoclonal anti-JNK (Proteintech), (xii) mouse monoclonal phospho-SAPK/JNK (Thr183/Tyr185, CST), (xiii) mouse monoclonal anti-caspase-8 (Proteintech), (xiv) rabbit polyclonal anti-caspase-3 (Proteintech), (xv) rabbit polyclonal anti-TNFR1 (Proteintech), and (xvi) mouse monoclonal anti-β tubulin as internal control (Abways Biotechnology, Shanghai, China).

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Immunoprecipitation, Microarray, Fluorescence

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: List of Immune-related Molecules of Human Macrophages Analyzed by Flow Cytometry

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), FITC-anti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421 TM -anti mouse CD11b (clone: M1/70), Brilliant Violet 570 TM -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/CF594-anti 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:

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: A CD73 inhibitor, AB680, showed a potent synergistic effect to improve therapeutic efficacy of a CDK4 inhibitor, palbociclib for CRC. ( A ) Representative histograms and ( B ) quantitative measurements of 50 μM ATP-induced PD-L1, CD39, and CD73 expression on macrophages, and the effects of treatment with 2 μM AB680 and CPI-444 (ciforadenant, 2 μM) from 5 independent experiments. ( C ) Relative level of cAMP in human PBMC-derived macrophages treated with or without ATP and AB680. ( D ) Quantitative measurements of TNF-α and IL-10 expression on macrophages treated with or without 2 μM AB680 and CPI-444 in the presence of 50 μM ATP and 100 nM LPS, from 5 independent experiments, using ELISA. ( E ) Outline of the AOM/DSS mouse model of CRC used in this study. ( F ) Representative images of large intestine and ( G ) graphical representation of number of tumors per AOM-DSS mouse administered with AB680 (20 mg/kg) or palbociclib (20 mg/kg) (n = 8 mice per group). ( H ) Representative histograms; and ( I ) quantification for PD-L1 level of TAMs in AOM-DSS mice administered with AB680 (20 mg/kg) or palbociclib (20 mg/kg) (n = 8 mice per group). ( J ) Time courses of tumor volumes in CT26 tumor-bearing mice administered with AB680 (20 mg/kg) or palbociclib (20 mg/kg) (n = 9 mice per group). Pooled data from in vivo experiments (n = 9 mice per group) showing mean fluorescence intensities (MFIs) of PD-L1 on intratumoral macrophages ( K ) percentage of TNFα-producing TAMs relative to intratumoral TAMs ( L ) and percentage of IL-10-producing TAMs from control, AB680-, palbociclib-, and AB680 plus palbociclib-administered tumor-bearing mice (n = 6 per group) ( M ). Data have been presented as mean ± standard error of the mean. ++ P < .01 AB680-treated vs vehicle-treated group; ## P < .01 palbociclib-treated vs vehicle-treated group; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001 AB680 plus palbociclib-treated vs vehicle-treated group (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), FITC-anti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421 TM -anti mouse CD11b (clone: M1/70), Brilliant Violet 570 TM -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/CF594-anti 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: Drug discovery, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Fluorescence, Control

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: Negative correlation between ecto-enzymes and CCND1 expression in CRC. ( A ) IHC staining of human normal colon (n = 40) and colon adenocarcinoma (n = 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 expression 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), FITC-anti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421 TM -anti mouse CD11b (clone: M1/70), Brilliant Violet 570 TM -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/CF594-anti 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: Nature Biotechnology

Article Title: The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models

doi: 10.1038/nbt.1665

Figure Lengend Snippet: Microarray data sets used for model development and validation in the MAQC-II project

Article Snippet: Raw microarray intensity data, instead of ratio data, were provided for MAQC-II data analysis NIEHS C Liver toxicants vs. non-toxicants based on overall necrosis score (rat) Affymetrix Rat 230 2.0 214 79 135 0.58 204 78 126 0.62 Exploratory visualization of the data set was reported in 2008 (ref. 53).

Techniques: Microarray, Generated, Positive Control, Negative Control

Journal: American Journal of Translational Research

Article Title: CHD1L promotes cell cycle progression and cell motility by up-regulating MDM2 in breast cancer

doi:

Figure Lengend Snippet: Identification of CHD1L target genes and network. A. The mRNA expression level and protein level of CHD1L in breast cancer cell lines were detected by RT-PCR (GAPDH was used as internal control) and western blot (β-actin was used as a loading control). B. Ectopic expression of CHD1L was detected in CHD1L-transfected cells by western blot (β-actin was used as a loading control). C. Left: Heatmap of the cDNA microarray analysis comparing the expression profiles between MDA-231 cells transfected with shCHD1L or control vector. Right: The up-regulated and down-regulated genes number in CHD1L-knockdown MDA-231 cells compared with control-231 cells. D. The top ten pathways regulated by CHD1L according to the P values of pathway enrichment analysis basing on differently expressed genes. E. The protein levels of CHD1L, MDM2, p53 were detected in Con-231, shCHD1L-231, Vec-474 and CHD1L-474 cells by Western blot analysis. β-actin was used as a loading control.

Article Snippet: Antibodies CHD1L (rabbit, HPA028670, sigma), β-actin (rabbit monoclonal, 12620s, Cell Signaling Technology, CST), MDM2 (rabbit, AF1244, R&D), p53 (mouse monoclonal, sc-126, Santa Cruz).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Microarray, Plasmid Preparation

Journal: American Journal of Translational Research

Article Title: CHD1L promotes cell cycle progression and cell motility by up-regulating MDM2 in breast cancer

doi:

Figure Lengend Snippet: CHD1L promotes tumorigenesis in nude mice through regulation of MDM2. IHC staining for CHD1L and MDM2 on serial sections of tumors induced by (A) Vec-474 and CHD1L-474 and (B) Con-231 and shCHD1L-231 cells. (100× magnification).

Article Snippet: Antibodies CHD1L (rabbit, HPA028670, sigma), β-actin (rabbit monoclonal, 12620s, Cell Signaling Technology, CST), MDM2 (rabbit, AF1244, R&D), p53 (mouse monoclonal, sc-126, Santa Cruz).

Techniques: Immunohistochemistry