Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

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: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: White matter microglia/macrophages are activated in the dorsal funiculi of male Plp1 mutant mice. Cervical spinal cord sections from ( A ) mildly-affected P16 rsh ( B ) severely-affected P16 msd ( C ) subclinical P25 Plp1 #66 heterozygous ( D ) moderately-affected P25 Plp1 #66 homozygous mice labeled with antibodies against Iba-1 (red) and CD68 (green). Many Iba-1 positive microglia/macrophages with swollen cell bodies and short processes are labeled with anti-CD68 antibodies in rsh and msd mice, which is indicative of activated cells. The insets in these panels show the canonical activated morphology revealed with anti-Iba-1 antibodies and strong CD68 staining. In contrast, there is little evidence for broad CD68 labeling in Plp1 #66 heterozygous mice in panel C, which is consistent with previous data that immune cells are rarely activated . Activation of microglia/macrophages is apparent for Plp1 #66 homozygous mice in panel D, which exhibit an intermediate level of CD68 labeling. Scale bar: 95 μm, insets 15 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Mutagenesis, Labeling, Staining, Activation Assay

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Proportion of cervical spinal cord Iba-1 + microglia/macrophages in dorsal column, ventral column and lateral funiculus that are activated (CD68 + ).

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques:

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Oligodendrocytes in gray matter from P16 rsh and msd mice do not undergo a UPR and microglia/macrophages exhibit resting morphology. ( A – C ) Iba1 (red) and CD68 (green) antibody labeling in dorsal spinal cord white matter tracts of P16 wild type ( A ) rsh ( B ) and msd ( C ) mice. Although the morphology and CD68 staining is characteristic of resting microglia/macrophages in wild type mice (white arrowheads), these cells are activated in the Plp1 mutants. ( D – F ) In contrast to white matter regions, microglia/macrophages have a resting state phenotype in the adjacent substantia gelatinosa (gray matter) from wild type and mutant mice (black arrowheads). ( G – I ) A major difference between these regions in rsh and msd mice is the relative abundance of oligodendrocytes undergoing an unfolded protein response (UPR), which is evident by the large number of CHOP + cells (green) in white matter (above the dotted line) compared to gray matter (below). We have previously shown that 100% of CHOP + cells in these mutants are oligodendrocytes . Dotted lines mark the white/gray matter boundary. Scale bar in I: 100 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Antibody Labeling, Staining, Mutagenesis

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Activation of rsh and msd microglia/macrophages in optic nerve at P16. ( A ) Longitudinal section from wild type mouse reveals Iba-1 + microglia/macrophages (red) with non-activated morphology (arrowheads). Most of these cells do not express CD68 or express the protein at low levels (green). ( B ) DAPI staining showing the nuclei of the microglia/macrophages in ( A ). ( C , E ) Iba-1 + microglia/macrophages from rsh ( C ) and msd ( E ) mice exhibit an activated morphology with enlarged cell bodies and thickened processes. Most of these cells express CD68 at high levels, which is localized to perinuclear regions. ( D , F ) DAPI staining of the fields in ( C , E ). Scale bar in F: 30 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Activation Assay, Staining

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Expression fold changes of other non-chromosome 17 interferon-induced genes in microarray data for rsh and msd mice.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Expressing, Microarray

Journal: PLoS ONE

Article Title: Functional Genomic Analyses of Two Morphologically Distinct Classes of Drosophila Sensory Neurons: Post-Mitotic Roles of Transcription Factors in Dendritic Patterning

doi: 10.1371/journal.pone.0072434

Figure Lengend Snippet: To access differences in gene expression, RNA from enriched C-I and C-IV neuronal populations were compared with that of whole larvae in microarray experiments. Live confocal images of neuronal populations labeled by ( A ) GAL4 221 (C-I) and ( B ) GAL4 ppk1.9 (C- IV) visualized by the trans-membrane fusion construct mCD8::GFP. Neurons have been pseudo-colored to distinguish individual subtypes and dendritic territories. ( C ) GAL4 221 strongly labels C-I da neurons along with weakly labelling of C-IV neurons in the background (dotted red trace). ( D ) GAL80 driven by a ppk promoter was combined in the background of GAL4 221 (ppk-GAL80; GAL4 221 ) that results in highly class I specific GAL4 expression. ( E ) A representative whole larval image of GAL4 ppk1.9 driving the expression of UAS-mCD8::GFP (gut is auto-fluorescent). ( F-J ) Strategy of class-specific neuronal isolation. Larvae expressing mCD8::GFP under the control of either ppk-GAL80; GAL4 221 or GAL4 ppk1.9 ( F ) were dissociated ( G ) filtered and incubated with superparamagnetic beads coated with anti-mCD8 antibody ( H ). The C-I/C-IV neurons bound to the magnetic beads were purified using a strong magnet ( I ), washed several time and used to perform microarray gene expression profiling ( J ). An identical region from the C-I and C-IV microarray are represented to show their dramatic qualitative differences ( J ). The microarray replicates were highly correlated, as represented in the correlation map ( K ). Principle component analysis revealed the three microarray samples from C-I, C-IV and whole larval lysate cluster into three distinct and well-defined clusters ( L ).

Article Snippet: The filtrate is then incubated with superparamagnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) coupled with biotinylated mouse anti-CD8a antibody (eBioscience) for 60 minutes.

Techniques: Gene Expression, Microarray, Labeling, Membrane, Construct, Expressing, Isolation, Control, Incubation, Magnetic Beads, Purification