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: 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: The Journal of Biological Chemistry

Article Title: Synthesis of Mitochondrial DNA Precursors during Myogenesis, an Analysis in Purified C2C12 Myotubes *

doi: 10.1074/jbc.M112.441147

Figure Lengend Snippet: Microarray analysis of genes regulating the dNTP pools in muscle cells. Samples were as follows: myoblasts ( Mb ), proliferating C2C12 myoblasts at 50% confluence; Myt , myotubes purified at 8 days of differentiation; EDL , extensor digitorum longus; and soleus ( SOL ), skeletal muscles dissected from adult CD1 mice. To measure expression levels, Cy3-labeled target from myoblasts, extensor digitorum longus and soleus preparations were hybridized in triplicate experiments on whole mouse genome oligonucleotide microarrays (4 × 44,000, Agilent Technologies). Normalized values were converted to log2 (scale on the left ), and heat maps were constructed with MultiExperiment Viewer to visualize gene expression levels. All genes involved in dNTP metabolism were annotated on the basis of their cytosolic or mitochondrial localization ( supplemental Table 1 ). To evaluate the significance of the expression changes, pairwise Significance Analysis of Microarrays two class analyses were carried out between myoblasts and each of the other samples. A , genes differentially expressed in at least one test using a false discovery rate of 1%. B , genes that showed no significant changes in muscle fibers compared with myoblasts. C , genes from A , sorted according to fold-change values to underline the predominant down-regulation observed in comparisons between cycling and differentiated muscle cells. A double asterisk marks genes with the most significant changes (false discovery rate, 0% in at least two tests). The inset shows the few genes with positive fold-changes.

Article Snippet: Incubation proceeded for 17 h at 65 °C at 10 rpm in a microarray hybridization oven (Agilent Technologies).

Techniques: Microarray, Purification, Expressing, Labeling, Construct

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