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: Nucleic Acids Research

Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing

doi: 10.1093/nar/gku818

Figure Lengend Snippet: Highly sensitive preparation of a sequencing library from 25 pg of ChIP DNA using TELP. ( A ) Validation of the sequencing libraries by qPCR. Histone H3K4me3 levels at nine genomic regions were determined through qPCR for sequencing libraries prepared using either the standard Illumina protocol with 10 ng ChIP DNA (Illumina 10 ng) or using TELP with the indicated amounts of ChIP DNA. The data were normalized to the promoter of the Cebpa gene (positive control, PC_1). The details of the qPCR primers are given in the Materials and Methods section. These results are the averages of three independent qPCR experiments, and the error bars indicate standard deviations. ( B ) A comparison of the H3K4me3 ChIP-seq signals from 10 ng ChIP DNA using the standard Illumina library construction method with TELP ChIP-seq analyses from 1 ng, 100 pg and 25 pg ChIP DNA. The data from a similar ChIP experiment using rabbit IgG were included as a negative control. ( C ) Scatter plots showing the comparison of the H3K4me3 levels (averaged RPKM signal intensities at the called H3K4me3 peaks, based on Illumina 10 ng) between those generated by the standard Illumina protocol and those generated by TELP using various amount of ChIP DNA. Pearson correlation coefficients, R , are indicated. ( D ) Venn diagrams showing overlaps between H3K4me3-marked regions identified by Illumina ChIP-seq (10 ng DNA) and TELP ChIP-seq (1 ng, 100 pg and 25 pg DNA). As significantly more peaks were called from the 25 pg DNA library due to elevated background noise ( n = 28,314), we selected those strong H3K4me3 peaks ( n = 10,733) by setting an additional selection criteria (Z-score normalized RPKM > 0) for this analysis.

Article Snippet: In parallel, 10 ng of the same ChIP DNA was used as starting material for the standard Illumina protocol for comparison.

Techniques: Sequencing, Biomarker Discovery, Positive Control, Comparison, ChIP-sequencing, Negative Control, Generated, Selection

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