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 Korean Medical Science

Article Title: Prediction of Microbial Infection of Cultured Cells Using DNA Microarray Gene-Expression Profiles of Host Responses

doi: 10.3346/jkms.2012.27.10.1129

Figure Lengend Snippet: The microarray experimental design in three dimensional spaces according to source of infection (x axis), cell type (y axis) and day of culture (z axis).

Article Snippet: Experiments were performed using the microarray system (Oligo-Human 10K, Macrogen Inc., Seoul, Korea) according to the manufacturer's protocol.

Techniques: Microarray, Infection

Journal: Gynecologic oncology

Article Title: Proteomic analysis of stage I endometrial cancer tissue: Identification of proteins associated with oxidative processes and inflammation

doi: 10.1016/j.ygyno.2011.02.031

Figure Lengend Snippet: Distribution of select differentially expressed proteins from the cancer versus normal endometrium comparison (ANXA1, ANXA2, PRDX1, PRDX3, PRDX5, PRDX6, COX2, COX6C, prohibitin 2, RL18 and RS18). P-values are shown for the Wilcoxon two-sample test (W) and for the Fisher exact test on the 2 by 2 presence/absence table (F). Each box extends from first to third quartile. Dark horizontal line is the median. The whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the box.

Article Snippet: Blots were washed again in 1X TBST followed by incubation in chemiluminescent substrate (SuperSignal West Pico, Pierce) for 5 min. Antibody and secondary antibody (2nd) conditions utilizedwere as follows: goat anti-rabbit IgGHRP (Abcam: ab6721), goat anti-mouse IgG-HRP (Pierce), anti-human GAPDH(Santa Cruz: sc-47724; 2nd at 1:100 K), anti-human annexin A1 (Cell Signaling: 3299; 2nd at 1:10 K), anti-Annexin A2 (Abcam: ab54771; 2nd: 1:100 K), anti-human lactate dehydrogenase (Abcam: ab55433; 2nd: 1:50 K) and anti-human peroxiredoxin 3 (Abcam: ab16753; 2nd at 1:50 K).

Techniques: Comparison

Journal: Gynecologic oncology

Article Title: Proteomic analysis of stage I endometrial cancer tissue: Identification of proteins associated with oxidative processes and inflammation

doi: 10.1016/j.ygyno.2011.02.031

Figure Lengend Snippet: A) Western blot analysis used to provide internal validation of normal postmenopausal endometrium and endometrial cancer tissue. B) Mean densitometric abundance levels of lactate dehydrogenase, annexin A1 and A2 and peroxiredoxin 3 derived from Western blot analyses of two normal postmenopausal endometrium and twelve endometrial cancer tissue samples. All values are normalized to GAPDH levels determined for each of these samples to control for loading.

Article Snippet: Blots were washed again in 1X TBST followed by incubation in chemiluminescent substrate (SuperSignal West Pico, Pierce) for 5 min. Antibody and secondary antibody (2nd) conditions utilizedwere as follows: goat anti-rabbit IgGHRP (Abcam: ab6721), goat anti-mouse IgG-HRP (Pierce), anti-human GAPDH(Santa Cruz: sc-47724; 2nd at 1:100 K), anti-human annexin A1 (Cell Signaling: 3299; 2nd at 1:10 K), anti-Annexin A2 (Abcam: ab54771; 2nd: 1:100 K), anti-human lactate dehydrogenase (Abcam: ab55433; 2nd: 1:50 K) and anti-human peroxiredoxin 3 (Abcam: ab16753; 2nd at 1:50 K).

Techniques: Western Blot, Biomarker Discovery, Derivative Assay, Control

Journal: Gynecologic oncology

Article Title: Proteomic analysis of stage I endometrial cancer tissue: Identification of proteins associated with oxidative processes and inflammation

doi: 10.1016/j.ygyno.2011.02.031

Figure Lengend Snippet: Densitometric results from Western blot validation of lactate dehydrogenase, annexins A1 and A2 and peroxiredoxin 3.

Article Snippet: Blots were washed again in 1X TBST followed by incubation in chemiluminescent substrate (SuperSignal West Pico, Pierce) for 5 min. Antibody and secondary antibody (2nd) conditions utilizedwere as follows: goat anti-rabbit IgGHRP (Abcam: ab6721), goat anti-mouse IgG-HRP (Pierce), anti-human GAPDH(Santa Cruz: sc-47724; 2nd at 1:100 K), anti-human annexin A1 (Cell Signaling: 3299; 2nd at 1:10 K), anti-Annexin A2 (Abcam: ab54771; 2nd: 1:100 K), anti-human lactate dehydrogenase (Abcam: ab55433; 2nd: 1:50 K) and anti-human peroxiredoxin 3 (Abcam: ab16753; 2nd at 1:50 K).

Techniques: Western Blot, Biomarker Discovery

Journal: Gynecologic oncology

Article Title: Proteomic analysis of stage I endometrial cancer tissue: Identification of proteins associated with oxidative processes and inflammation

doi: 10.1016/j.ygyno.2011.02.031

Figure Lengend Snippet: Representative immunohistochemical staining of ANXA2 (A) and PRDX1 (B) in cancer (left panels, A1 and B1) and normal postmenopausal endometrium (right panels, A2 and B2) in a tissue microarray. (C): Boxplots for tissue microarray blinded pathologic assessments for annexin A2 (ANXA2) and peroxiredoxin (PRDX1), taking on values 0, 1, 2, and 3. The boxes extend to first and third quartiles, thick lines are at the median, and whiskers extend to 1.5× width of box or to the range of the data. The P-value for ANXA2 is from analysis of variance. The P-value indicated by (A) is from a Wilcoxon test normalizing for total sample spectral count.

Article Snippet: Blots were washed again in 1X TBST followed by incubation in chemiluminescent substrate (SuperSignal West Pico, Pierce) for 5 min. Antibody and secondary antibody (2nd) conditions utilizedwere as follows: goat anti-rabbit IgGHRP (Abcam: ab6721), goat anti-mouse IgG-HRP (Pierce), anti-human GAPDH(Santa Cruz: sc-47724; 2nd at 1:100 K), anti-human annexin A1 (Cell Signaling: 3299; 2nd at 1:10 K), anti-Annexin A2 (Abcam: ab54771; 2nd: 1:100 K), anti-human lactate dehydrogenase (Abcam: ab55433; 2nd: 1:50 K) and anti-human peroxiredoxin 3 (Abcam: ab16753; 2nd at 1:50 K).

Techniques: Immunohistochemical staining, Staining, Microarray