A simple technique for quantifying apoptosis in 96-well plates

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.

<p>Figure S1. Oligonucleotide pairs containing portions of human JNK1 and JNK2 were used to create retroviral vectors for delivery of shRNAs into HT29 cells. Target sequences (underlined) were taken from Hui et al ( J Clin Invest, 2008,118:3943-53). Oligonucleotide pairs were synthesized, annealed at equimolar concentration, diluted and ligated into pSUPER-RetroPuro-DR, a modified form of pSUPER-RetroPuro (OligoEngine, Seattle, WA). The resulting vectors were transfected into Phoenix-Ampho packaging cell line, kindly provided by Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA) using Fugene 6 reagent (Roche). Viruses were collected, purified, aliquoted and stored at -80oC. Figure S2. Functional JNK1 in necessary for induction of autophagy in HT29 cells. Cells were subjected to hypoxia and oxaliplatin (5 mM) for 24 hours. Acridine orange (AO) staining was performed as follows: cells were washed twice with PBS, stained with 1 μm/ml acridine orange for 15 min at 37 {degree sign}C, washed again thrice and observed under microscope in PBS solution. Custom light cube for AO staining was used with blue (ca. 450 nm) excitation and red (ca. 650 nm) emission filters. Under AO staining, the cytoplasm and nucleus fluoresce green, whereas the acidic compartments fluoresce bright red or orange-red Our results show lesser content and size of acidic vesicles (including autolysosomes) in HTJ1.3 cells, as compared to control and HTJ2.2 lines. Scale bar - 100 mm. Figure S3. Results of MTT assays in HT29-derived cell lines after retroviral introduction of shRNA for JNK1 (sJ1) or JNK2 (sJ2). Shown are the average values of IC50 derived from three independent experiments in triplicate. Bars represent standard deviation; ***, P <0.001. Figure S4. Apoptosis is maximally enhanced by hypoxia and CQ in the HTJ1.3 cell line. A, Cells were seeded on glass slides and subjected to hypoxia for 24 hours with or without chloroquine (3 mM), followed by staining using Apoptosis and Necrosis Quantification Kit (Biotium). Apoptotic cells were labeled with FITS-Annexin V (green), necrotic - with Ethidium Homodimer III (red). The whole width of each image corresponds to 100 mm. B, Apoptosis was quantified by manual count of stained cells in several fields (n=5). Graph represents the percentage of cells stained with Annexin. EtBr-stained nuclei showed lower count and almost always coincided with Annexin stain (suggesting late apoptosis), and only showed slighter higher count (1-2% more cells with only EtBr staining) where marked by a dot. *, P<0.05. Figure S5. Following hypoxia/reoxygenation treatment (24+24 hours, 5 mM of oxaliplatin), autophagy still inhibited in JNK1-deficient cell line, but apoptosis is activated equivalently in all cell lines, most likely due to persistent stress, exacerbated by ROS formation, after oxygen supply is restored. Figure S6. Acridine orange staining of Bcl-XL-deficient HT29 cells shows slightly lower accumulation of acidic vesicles, when compared to parental cell line, earlier during hypoxia. By 24 hours of hypoxic exposure, however, this difference is no longer detectable. Scale bar - 100 mm. Table S1. Cytotoxic interactions between autophagy inhibitor (CQ) and JNK inhibitor (CC-401) in colon cancer cells.</p>

An in vitro comparison of two different subpopulations of retinal progenitor cells for self-renewal and multipotentiality

Most cultured cells used for biomedical research are cultured adherently, and the requisite detachment prior to biochemical analysis might induce chemical changes. This is especially crucial if accurate metabolic measurements are desired, given the rapid turnover of metabolites in living organisms. There are only a few methods available for the nontargeted in situ analysis of small adherent cell populations. Here we show that laser ablation electrospray ionization (LAESI) mass spectrometry (MS) can be used to analyze adherent cells directly, while still attached to the culture surface. To reduce the size of the analyzed cell population, the spot size constraints of conventional focusing in reflection geometry (rg) LAESI had to be eliminated. By introducing transmission geometry (tg) LAESI and incorporating an objective with a high numerical aperture, spot sizes of 10-20 μm were readily achieved. As few as five adherent cells could be specifically selected for analysis in their culturing environment. The importance of in situ analysis was highlighted by comparing the metabolite composition of adherent versus suspended cells. For example, we observed that cells analyzed adherently yielded higher values for the adenylate energy charge (0.90 ± 0.09 for adherent cells vs 0.09 ± 0.03 for suspended cells). Additionally, due to the smaller focal spot size, tg-LAESI enabled the analysis of ∼20 times smaller cell populations compared to rg-LAESI.

The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.

When mononuclear cells from peripheral human blood were plated directly in agar medium containing phytohemagglutinin (PHA) and sheep red blood cells and cultured for 6–7 days, discrete colonies of 4–50 lymphocytes were formed. The plating efficiency was one colony per 50–100 cells plated, but this was reduced by approximately 85 % when adherent cells were removed from the mononuclear cell suspension prior to culture. PHA‐induced colony formation by autologous or homologous nonadherent cells could be completely restored by culturing nonadherent cells over a feeder layer of adherent cells or addition of the medium in which adherent cells had been cultured. Thus, adherent cells were presumed to secrete a stable soluble factor which supported PHA‐induced colony formation.This active factor in conditioned medium from adherent cells could be stored for several months at –10°C, and was relatively stable when heated to 56°C for 30 min. It did not bind to concanavalin A‐Sepharose, had a molecular weight greater than 10 000 daltons, was inactivated by treatment with pronase but not by DNAse, RNAse or lipase, and was active at a concentration of 1 %. Adherent cells and culture media from human peritoneal macrophages and fibroblasts did not stimulate colony formation in nonadherent cells, and likewise culture medium from nonadherent cells or purified T lymphocytes was inactive. When concentrates of conditioned medium from cultures of adherent cells were fractionated on a Sephadex G‐100 column two peaks of activity were obtained: a sharp peak with a molecular weight of approximately 100 000 daltons and a smaller peak with a molecular weight between 12 000 and 15 000 daltons.Our data indicate that a factor or factors of high molecular weight are produced by cultures of adherent mononuclear cells; these factors support the formation of PHA‐induced colonies by human T lymphocytes.

Effects of osmotic and cold shock on adherent human mesenchymal stem cells during cryopreservation

Anti-proliferative and apoptosis induction activities of extracts from Thai medicinal plant recipes selected from MANOSROI II database

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE(-/-) mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

Undertaker, a Drosophila Junctophilin, Links Draper-Mediated Phagocytosis and Calcium Homeostasis

Diagnosis of Trichomonas vaginalis infection: The sensitivities and specificities of microscopy, culture and PCR assay

Bone marrow (BM) consists of both hematopoietic cells and non-hematopoietic stromal cells, which support hematopoiesis. Recent reports have suggested that some non-hematopoietic BM cells, such as multipotent adult progenitor cells, can produce hematopoietic stem and/or progenitor cells. It has been speculated that non-hematopoietic BM stromal cells may generate myeloid malignancy following conversion of non-hematopoietic cells to hematopoietic cells. To study the conversion of non-hematopoietic cells (CD45-) to hematopoietic cells (CD45+), we used the NUP98-HOXD13 (NHD13) mouse model of myelodysplastic syndrome (MDS). NHD13 mice develop MDS which mimics the human condition in terms of peripheral blood (PB) cytopenias, ineffective hematopoiesis, and transformation to acute myeloid leukemia (AML). Two cell lines (251 and 63B) established from NHD13 mice with AML grow as a combination of adherent and suspension cells. Upon sub-culture of the suspension cells, the suspension cells become adherent. Although the suspension cells are almost entirely (>95%) CD45+, the adherent cells are only 50% CD45+, suggesting that a substantial fraction of the CD45+ cells became CD45-. In conjunction with acquisition of CD45, the cell lines acquire Mac-1, CD16/32 and F4/80 positivity, which are lost upon loss of CD45. In order to rule out the possibility that a small fraction of CD45- cells in the suspension phase (99.85%) population of CD45+ cells. After 14 days of sub-culture, FACS revealed that 46% of the cells were now CD45-, demonstrating conversion of CD45+ cells to CD45- cells. To further explore this finding, CD45+ and CD45- populations were purified and expression of the NHD13 transgene (driven by the pan-hematopoietic Vav1 promoter) was determined by Q-PCR to be 100-fold higher in the CD45+ fraction compared to the CD45- fraction. These results demonstrate that the cell lines can cycle between non-hematopoietic and hematopoietic cells. To determine if the cell lines have retained malignant potential, we transplanted irradiated recipients via intravenous injection. One of 3 recipients transplanted with 1×10^6 cells showed low level engraftment [1.7% of peripheral blood (PB) cells] at week 24 post-transplant. Remarkably, this mouse was leukemic 2 weeks later, with splenomegaly, anemia, and monocytosis (8.57 K/uL), and donor engraftment of 17-53% in the PB, BM, and spleen. To improve engraftment, we used intra-femoral injection, which resulted in consistent engraftment at 6 weeks in the PB. This experiment is ongoing; at 9 weeks one recipient has developed lymphopenia and anemia, suggesting the possibility of malignant transformation. Taken together, these findings suggest that malignant cells can cycle between stromal and hematopoietic cells, and support the hypothesis that non-hematopoietic BM cells may be an important part of the malignant clone in patients with MDS and AML. Citation Format: Yang Jo Chung, Suntae Kim, Peter Aplan. De-differentiation of CD45+ hematopoietic cells to CD45- stromal cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5086. doi:10.1158/1538-7445.AM2015-5086

A comparison of acridine orange, gram stain and culture for the detection of organisms in ocular specimens