Journal: The Journal of Biological Chemistry

Article Title: Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1 *

doi: 10.1074/jbc.M113.480517

Figure Lengend Snippet: qPCR validation of LCA microarray analysis and changes in gene expression caused by treatment with control compound, anthraquinone-2-carboxylic acid

Article Snippet: Microarray Analysis Total RNA was isolated from 2 × 10 5 cells and used for microarray analysis (University of Iowa DNA Core Facility) in hybridization to Human Gene ST1.0 Array GeneChips (Affymetrix).

Techniques: Biomarker Discovery, Microarray, Gene Expression, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 2. Upregulation of CXCL10 and MHC class II in human neutrophils in urine during BCG infusion therapy. (a,b) Comprehensive analysis of mRNA expression in urine-derived neutrophils compared to peripheral blood neutrophils. Blood and urine were collected from three patients after one week from the 6th BCG infusion. Comprehensive analysis of mRNA in neutrophils was performed using a DNA tip microarray. (a) Cluster analysis after adjustment and standardiza- tion. The mRNA expression in neutrophils obtained from urine samples (vertical axis) or periph- eral blood (horizontal axis) was analyzed. White lines indicate the thresholds for genes that are upregulated or downregulated > 2-fold between urine- and blood-derived neutrophils. A relatively higher expression in urine-derived neutrophils is indicated using arrows, including expression for CXCR3 ligands (CXCL9 and CXCL10) and MHC class II (HLA-DRB1, HLA-DPA1, and HLA-DQA1). (b) Volcano plot depicting the differentially expressed genes between peripheral blood-derived and urine-derived neutrophils after the 6th BCG infusion. The horizontal axis denotes the fold change in mRNA expression in neutrophils from the urine and blood, while the vertical axis represents the –log10 (p-value) for a t-test of differences in neutrophils from the blood and urine. These data represent the top 6000 genes of the –log10 (p-value). The gene expressions of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) and MHC class II (HLA-DQA2, HLA-DPA1, and HLA-DQA1) were also detected as characteristic features of urine-derived neutrophils (arrows). (c,d) Representative data of intracellular- stained neutrophilic cells obtained via flow cytometric analysis. The CD33+CD15+ neutrophilic cells in the blood (c) or urine (d) samples were obtained from the same patient who was treated with 4th BCG infusions and are presented as CXCL10 MFI (upper panels) and HLA-DR MFI (lower panels). Gray-closed histograms indicate each background staining, and light blue line histograms denote the staining of CXCL10 or HLA-DR. (e–g) Comparison of intracellular expression of (e,f) CXCL10 and (g) HLA-DR in neutrophilic cells from the blood (open circle) and urine (closed circle) samples. These samples were collected after one week from the 2nd to the 6th BCG infusions (after each infusion). (e) ∆CXCL10 MFI was calculated as follows: ∆CXCL10 MFI = (MFI of PE-conjugated anti-CXCL10 mAb staining) −(MFI of PE-conjugated control IgG staining). (f) The neutrophilic cells

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Derivative Assay, Microarray, Staining, Comparison, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 3. Effect of BCG on CXCL10 and MHC-II expression in human or mice neutrophilic cells in vitro. Human (a,d) or mouse (b,e) peripheral blood was diluted ten-fold in 10% FCS RPMI1640, or mouse bone marrow cells (4 × 106/mL; c,f) were incubated with or without 4 µg/mL of BCG for 20 h. Following incubation, the expression levels of CXCL10 (a–c) and MHC class II (d–f) in human (CD33+CD15+) or mouse neutrophils (CD45+Ly6G+) were analyzed, as described in Figure S2. Statistical significance was calculated with the paired t-test, * p < 0.05 (n = 3). Abbreviations: BCG, Bacillus Calmette–Guérin; CXCL10, chemokine (C-X-C motif) ligand 10; HLA-DR, human major histocompatibility complex class II cell surface receptor; MFI, mean fluorescence intensity; and I-A/I-E, mouse major histocompatibility complex class II cell surface receptor.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, In Vitro, Incubation, Immunopeptidomics, Cell Surface Receptor Assay

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 4. Upregulation of CXCL10 and MHC class II in monocytes and neutrophils in peritoneal effusion cells after BCG injections. Mice were injected with B16F10 cells (5 × 104 cells/100 µL/head), and the PECs were collected after two weeks. The PECs induced after one injection of BCG (40 µg/head) after 16 h and the PECs induced after five repeated injections of BCG (40 µg/head) after 16 h from the final injection are presented as “1-shot” and “5-shots”, respectively. These PECs were intracellularly stained with each antibody, and the relative expression (MFI) of CXCL10 and I-A/I-E was analyzed in CD45+Ly6C+ cells and CD45+Ly6G+ cells, respectively. (a–i) Representative flow cytometric analysis of mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) via flow cytometry. The (a–c) panels present flow cytometric analysis of the PECs induced 2 weeks after B16F10 cell injection (presented as “Tumor”). The (d–f) panels show representative flow cytometric analysis of the PECs induced 16 h after the administration of BCG (presented as “1-shot). The (g–i) panels indicate representative flow cytometric analyses of the PECs induced via five repeated BCG injections at one-week intervals. The PECs were collected 16 h after the final BCG admin- istration (presented as “5-shots”). The left panels (a,d,g) show CD45+ leukocytes presented with the gates of Ly6C+ cells (monocytic cells) and Ly6G+ cells (neutrophilic cells). (j–l) The number and proportion of myeloid cells (Ly6C+ and Ly6G+ cells) of the PECs. The peritoneal effusion cells obtained after injection of B16F10 cells are presented as “tumor” (open circles). The cells induced 16 h after a single administration of BCG are presented in the group “1-shot” (closed circles). The cells induced via five repeated injections of BCG are presented in the group “5-shots” (closed triangles). The (j) number of the cells in peritoneal fluid were counted using a hemocytometer, and the proportions of (k) Ly6C+ cells and (l) Ly6G+ cells in CD45+ leukocytes were analyzed via flow cytometry. (m–p) The intracellular expression levels of CXCL10 and MHC-II (I-A/I-E) in mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) after BCG injection. These PECs were intracellularly stained with each anti- body, and the relative expression (MFI) of (m,n) CXCL10 and (o,p) I-A/I-E was analyzed in (m,o) CD45+Ly6C+ cells and (n,p) CD45+Ly6G+ cells, respectively. Statistical analyses were per- formed using the Kruskal–Wallis test with the Dunn’s post-hoc test. Each bar is presented as the mean of data. * p < 0.05; ** p < 0.01; and ns, not significant. Abbreviations: PECs, peritoneal exudate cells; CXCL10, C-X-C motif chemokine ligand 10; BCG, Bacillus Calmette–Guérin; and MFI, mean fluorescence intensity.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Injection, Staining, Expressing, Cytometry

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 7. CXCL10 and MHC class II expression in neutrophils induced via BCG was inhibited via partial neutrophil depletion using anti-Ly6G mAbs. BCG (40 µg/100 µL/head) was injected into the peritoneal cavity, following which the antibodies (100 µg/50 µL/head; control mAb, open circle; or anti-Ly6G mAb, closed circle) were injected into the

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Injection, Control

Journal: Molecular Therapy

Article Title: Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

doi: 10.1038/mt.2013.210

Figure Lengend Snippet: Expression of Caveolin 1 and 2 correlates with efficient siRNA transfection with alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs). (a) Transfection efficiency of three tested leukemia cell lines, K562 (easily transfected), Molm13 (modestly transfected) and KG1 (poorly transfected), correlates with the amount of siRNAs entering into cells. Cy3-labeled control luciferase siRNAs were transfected into K562, Molm13 and KG1 cells using SLP301R. The cellular entry of siRNA was measured by quantitative fluorescent imaging (ImageStreamX, Amnis) at 2 hours after transfection. Mean Cy3 florescent intensity with SD was shown on the left. Two representative images from each cell line were shown on the right. (b) Four endocytosis-related genes were identified to be significantly underexpressed in poorly transfected KG1 and Mv4-11 cells compared with modestly transfected Molm13 and THP1 cells by comparative microarray analysis (Supplementary Table S2). (c) The expression levels of candidate genes identified in microarray were confirmed by quantitative RT-PCR in cell lines as indicated. Cav1, Cav2, and Rab13 were confirmed as underexpressed genes in poorly transfected KG1 and Mv4-11 cells compared with modestly and easily transfectd cell lines Molm13, THP1, HEL, and K562. (d) Upper panel, three groups of cell lines including easily transfected and poorly transfected adherent cell lines as well as hardest-to-transfect suspension leukemia cells, were subjected to comparative microarray analysis (Supplementary Tables S3 and S4). Lower panels, Cav1 and Cav2 were confirmed by quantitative RT-PCR as overexpressed genes in easily transfected adherent cell line HCT116, as compared with poorly transfected adherent cell lines HT29 and Colo205, and suspension leukemia cell line K562. (e) Colocalization of siRNA and Caveoloae. Cy3-labeled control luciferase siRNAs encapsulated in SLP301R were coadministered into K562 cells with Alexa647-labeled Albumins, which have been known to enter cells through Caveolae-mediated endocytosis. The cellular entry of siRNAs and Albumins was measured by quantitative fluorescent imaging (ImageStreamX, Amnis) at 30 minutes after administration. Two representative colocalization images were shown.

Article Snippet: The human adherent cancer cell lines, PC3, Colo205, HCT116, and HT-29 (ATCC, Manassas, VA); human leukemia cell lines, MV-4;11, K562, KG1, HEL, THP1 (ATCC); and MOLM13 (DSMZ, Braunschweig, Germany), were maintained in corresponding media (DMEM for adherent cell lines and RPMI for leukemia cell lines) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen).

Techniques: Expressing, Transfection, Labeling, Control, Luciferase, Imaging, Microarray, Quantitative RT-PCR, Suspension

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: Antibodies used in this study include: CRABP-II mouse mAbs (Millipore, MAB5488), CRABP-II rabbit polyclonal antibody (Proteintech, 10,225–1-AP), HuR (3A2, Santa Cruz, sc-5261), Flotilin-2 (Santa Cruz, sc-28320), GAPDH (Santa Cruz, sc-365062), and Actin (Santa Cruz, sc-1615), anti-Flag M2 mAb (Sigma, F9291), anti-Flag agarose beads (Clontech, #635,686), Ki67 (SP6, ThermoFisher, RM-9106-S0), ADRP (Novus, NB110-40,877), Caspas3 (Cell Signaling, #9662), PARP (Cell Signaling, #9542), AKT (Cell Signaling, #4691), mTOR (Cell Signaling, #2983), S6 (Cell Signaling, #2217), pAKT (S473, Cell Signaling, #9018), pmTOR (Cell Signaling, #5536), pS6 (Cell Signaling, #4858), and pGSK3β (Cell Signaling, #5558).

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A ) and ( B ) show Fluorescence-Activated Cell Sorting (FACS) results and the respective bar graphs. Error bars represent standard error of the mean (SEM). The p -values were calculated using an unpaired t test. * P < 0.05; *** P < 0.001. (A) The cells were treated with 6 μM docetaxel for 1 to 3 days and then stained with CD24-PE and CD44-FITC for FACS analysis. HCC1806, HCC1937 and HCC38 are TNBC cell lines. JIMT-1 is a HER2-overexpressing BC cell line. (B) HCC1806 and HCC1937 were treated with 4 μM doxorubicin for 1 to 3 days and then stained with CD24-Brilliant Violet 421 and CD44-FITC. ( C ) The summarized results of A and B.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques: Fluorescence, FACS, Staining

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A ) Cells were treated with 10 μM ATM inhibitor KU60019. ( B ) FACS results showed that 10 μM ATM inhibitor increased CD24 expression in MDA-MB-231 cells; and 4 μM doxorubicin reduced CD24 expression. ( C ) Selected cells were transfected with control vector, CD24 shRNA or NDRG2 shRNA. Knockdown of NDRG2 caused dramatic p-ATM increase in HCC1806 and MDA231 cells ( D ) The cells were treated with docetaxel 6.4 μM or doxorubicin 4 μM. Both docetaxel and doxorubicin increased p-TGF-βR1 in the three cell lines. Doxorubicin reduced p-Bcl-2 in HCC1937 and docetaxel elevated p-Bcl-2 in HCC1806 and HCC38.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, shRNA, Knockdown

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A ) The drug sensitivity assay results of six TNBC cell lines dosed with either 25.6 μM docetaxel or 6.4 μM doxorubicin are shown. P -values were calculated with Two-way ANOVA. * P < 0.05; *** P < 0.001. ( B ) After seeding in matrigel for 4 days, cells were treated with 0.6 μM docetaxel or 60 nM doxorubicin for 7 days. ( C ) HCC1937 cells were cultured under standard conditions or in ultralow-attachment plates for 2 days, then treated with 6 μΜ doxorubicin or 25.6 μΜ docetaxel for either 2- or 7 days. *** P < 0.001; **** P < 0.0001. ( D ) Work flow for experiments shown in ( E ) and ( F ). HCC1806 and HCC38 cells were treated with 25.6 μM docetaxel for 2-days. Treated cells were then sorted into CD44 + /CD24 +/high and CD44 + /CD24 −/low populations by FACS. Sorted populations were re-treated with 25.6 μM docetaxel, with cell viabilities determined after 2-days. MDA-MB-468 and HCC1937 cells underwent the same protocol except 6.4 μM doxorubicin was used. (E) and (F) P -values were calculated with unpaired t test. * P < 0.05; ** P < 0.01. Error bars represent SEM. Scale bar, 200 μm.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques: Sensitive Assay, Cell Culture

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A , B , C and D ) After seeding in matrigel for 5-days, HCC1806 cells were treated with 20 nM docetaxel (DTX) or 1.5 μM doxorubicin (DXR) for 4-days. HCC1937 cells were treated with 0.6 μM docetaxel. P -values of the compared groups were calculated using unpaired t -tests for cell total area. ( E ) These data show the working scheme for treatments used in tumor xenograft experiments. ( F , G , H ) These data show results of experiments with five tumors in each treatment group. ( I ) These data show statistical analyses of tumor shrinkage in the three pairs of experiments. P -values of each paired group were calculated with paired sample t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001. Error bars represent SEM. Scale bar, 200 μm.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques:

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A ) Results from three cell lines, HCC1937, HCC1806 and HCC38, treated with 6.4 μM docetaxel (DTX), 4 μM doxorubicin (DXR) or 5 μM STAT3 inhibitor VII. Levels of phospho-ATM, -NDRG2 and -STAT3 showed congruent changes following a similar trend: p-NDRG2 and p-STAT3 were increased when p-ATM was upregulated; p-NDRG2 and p-STAT3 were decreased when p-ATM was suppressed. ( B ) Gene microarray data show that expression of genes in the ATM pathway was dramatically reduced after 6 h of 4 μM docetaxel treatment. The genes changed ≥ 2 folds and P < 0.05.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques: Microarray, Expressing

Journal: Oncotarget

Article Title: CD24 Expression and differential resistance to chemotherapy in triple-negative breast cancer

doi: 10.18632/oncotarget.16203

Figure Lengend Snippet: ( A ) and ( B ) Western blot results. Docetaxel: 6.4 μM; doxorubicin: 4 μM; cyclophosphamide: 2 μM. The results showed that doxorubicin reduced p-Bcl-2 in HCC1937 and HCC1806 and docetaxel increased p-Bcl-2 in the same cell lines. Cyclophosphamide had no significant effect on p-Bcl-2 in the two cell lines. HCC38 had a relatively higher basal level of TGF-βR1 compared to HCC1937. The bands of p-Bcl-2, Bcl-2 and β-actin in HCC1806 treated with DTX and HCC1937 treated with DXR have been shown in Figure . They were shown here again for a different comparison. ( C ) Cells were treated with 5 μM Bcl-2 inhibitor ABT-737. The results showed that Bcl-2 inhibitor stimulated p-TGF-βR1 and p-ATM, suggesting that Bcl-2 is an inhibitor of TGF-βR1 and ATM in the three cell lines. ( D ) Cells were treated with 5 μM TGF-βR1 inhibitor LY 364947. The results showed that TGF-βR1 inhibitor suppressed p-ATM and TGF-βR1 is stimulatory to p-ATM. ( E ) The Western blot results of autophagy marker-LC3B. The results showed that CD24 knockdown increased LC3B expression and NDRG2 knockdown eliminated LC3B expression. ( F ) Proposed diagram to summarize contrasting effects of doxorubicin and docetaxel on critical TNBC cell signaling pathways.

Article Snippet: HCC1806 cells were treated with DMSO or 4 μM docetaxel for 6 h. After treatment, mRNAs of samples were prepared and submitted for Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, U.S.A.) analysis in duplicate.

Techniques: Western Blot, Comparison, Marker, Knockdown, Expressing, Protein-Protein interactions

Journal: Cell reports

Article Title: Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST

doi: 10.1016/j.celrep.2019.03.072

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-calbindin D-28K monoclonal antibody (clone CB-955) , Acris Antibodies , Cat# AM08219SU-N; RRID: AB_1954252.

Techniques: Expressing, Plasmid Preparation, Recombinant, SYBR Green Assay, Reporter Assay, Isolation, Staining, Microarray, Clone Assay, Software

Journal: Scientific reports

Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.

doi: 10.1038/s41598-018-31567-3

Figure Lengend Snippet: Figure 6. BA inhibits USP10 in LNCaP and 22Rv1. Western blot analysis of DUB labeling assay using HA- UbVS and USP10, 9X, and 7 specific antibodies. Results showed that BA inhibited USP10 activity (ratio of USP10/HA/total USP10 values shown below; 0 h = 1). BA inhibited USP9X activity in 22Rv1 but not in LNCaP. There was little effect of BA on USP7 activity. Blot images were cropped for clarity of the presentation. Similar results were obtained in an additional experiment.

Article Snippet: Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68.

Techniques: Western Blot, Labeling, Activity Assay

Journal: Scientific reports

Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.

doi: 10.1038/s41598-018-31567-3

Figure Lengend Snippet: Figure 7. USP10, a candidate AR-regulatory DUB inhibited by BA, is variably expressed in human PCa tissues. (a) Western blot showed that stable knockdown (5- to 14-fold) of USP10 in LNCaP with 3 different shRNAs (shUSP10-1, -2, -3) reduced AR protein by 1.5- to 5-fold compared to shGFP control. Stable overexpression of USP10 (6-fold) increased AR protein 2-fold compared to empty vector (EV) control. Loading control (protein). (b) In 22Rv1, stable knockdown of USP10 (6- to 100-fold) reduced AR and AR-V7 protein by 1.5- to 3-fold in 2 of 3 shRNAs. Blot images in (a,b) were cropped for clarity of the presentation. (c) Representative IHC images of USP10 expression (x200, dark brown color) in human PCa tissues compared to normal prostate using a tissue microarray. Results showed that USP10 was highly expressed in cytoplasm and nucleus of epithelial cells in normal prostate. With higher Gleason (G7 and 9 compared to G4) grades of PCa, USP10 was more variably expressed with less nuclear localization.

Article Snippet: Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68.

Techniques: Western Blot, Knockdown, Control, Over Expression, Plasmid Preparation, Expressing, Microarray

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDAC1, HDAC2, and HDAC3 were upregulated in breast cancer tissues and correlated with worse prognosis in breast cancer patients. ( a ) Representative immunohistochemical (IHC) staining photos of HDAC1, HDAC2, and HDAC3 in breast specimens. HDAC1, HDAC2, and HDAC3 levels were obviously elevated in the tumor tissues compared to the non‐neoplastic adjacent tissues of patients with invasive ductal carcinoma (IDC). Yellow boxes indicated mammary ducts. Scale bars, 100 μm. ( b ) Representative IHC photos of three different kinds of HDAC3 subcellular localization. Scale bars, 100 μm. ( c – f ) Overall survival (OS) curves of 139 IDC patients with different HDAC1 ( c ) ( ) HDAC1 Low ( ) HDAC1 High ( ) HDAC1 low‐censored ( ) HDAC1 high‐censored, HDAC2 ( d ) ( ) HDAC2 Low ( ) HDAC2 High ( ) HDAC2 low‐censored ( ) HDAC2 high‐censored, cytoplasmic HDAC3 ( e ) ( ) HDAC3 C‐low ( ) HDAC3 C‐high ( ) HDAC3 C‐low‐censored ( ) HDAC3 C‐high‐censored, and nuclear HDAC3 ( f ) levels ( ) HDAC3 N‐low ( ) HDAC3 N‐high ( ) HDAC3 N‐low‐censored ( ) HDAC3 N‐high‐censored. According to another set of criteria in which cases with either high nuclear or cytoplasmic expression were classified into the C‐high/N‐high group and other cases were classified into the Others group, the overall survival curve of the 139 IDC patients was reproduced as Figure 1g ( ) HDAC3 Others ( ) HDAC3 C‐high/N‐high ( ) HDAC3 Others‐censored ( ) HDAC3 C‐high/N‐high‐censored. P values of the Kaplan‐Meier plots in (c‐g) were calculated by log‐rank test in IBM SPSS Statistics 19 software. ( h – j ) Kaplan‐Meier survival curves with log‐rank analysis were used to assess the correlation between HDAC1 ( h ) ( ) HDAC1 Low (≤ 75th percentile) ( ) HDAC1 High (> 75th percentile), HDAC2 ( i ) ( ) HDAC2 Low (≤ 75th percentile) ( ) HDAC2 High (> 75th percentile), and HDAC3 ( j ) ( ) HDAC3 Low (≤ 80th percentile) ( ) HDAC3 High (> 80th percentile) expression and overall survival of 4903 breast cancer patients in the bc‐GenExMiner platform (website: http://bcgenex.centregauducheau.fr ; all DNA microarray data, node mixed, ER mixed; optimized split for HDAC1 and 2, an 80th percentile customized cutoff for HDAC3).

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Software, Microarray

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in overall survival of IDC patients ( n = 16l)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: Relationship between clinicopathological characteristics and HDACs expression in IDC patients ( n = 139)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Over Expression

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in the onset of brain metastasis of IDC patients ( n = 161)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: The roles of HDACs expression and other clinicopathological characteristics played in the prognosis of breast cancer patients after brain metastasis ( n = 63)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing