Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages

Noursadeghi, Mahdad; Tsang, Jhen; Haustein, Thomas; Miller, R F; Chain, Benjamin M.; Katz, David R.

doi:10.1016/j.jim.2007.10.015

Cited by 158 publications

(146 citation statements)

References 6 publications

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“…RELA translocation was quantified in 250 cells from 3 independent experiments as described. 65 LC3 and LC3-LysoTracker Red colocalized puncta per cell were determined using a green and red puncta colocalization macro 66 and ImageJ software in threshold images with sizes from 3 to 30 pixel 2 and puncta circularity 0.8 to 1 in more than 50 cells from 3 independent experiments scored in random fields.…”

Section: Western Blot Analysis Of Cell Lysatesmentioning

confidence: 99%

The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

et al. 2015

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Abbreviations: 3-MA, 3-methyladenine; AKT, v-akt murine thymoma viral oncogene homolog; ALB, albumin; ATG7, autophagyrelated 7; CD, cluster of differentiation; CD5L, CD5 molecule-like; FCS, fetal calf serum; FSL1, pam2CGDPKHPKSF; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; MAP1LC3A/B (LC3), microtubule-associated protein 1 light chain 3 a/b; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MK, macrophages; MAPK, mitogen-activated protein kinase; moAb, monoclonal antibody;; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; oxLDL, oxidized low-density lipoprotein; PB monocytes, peripheral blood monocytes; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; poAb, polyclonal antibody; PMA, phorbol 12-myristate 13-acetate; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; r-HsCD5L, recombinant human (Homo sapiens) CD5L; RELA, v-rel avian reticuloendotheliosis viral oncogene homolog A; siRNA, short interference RNA; SRCR, scavenger receptor cysteine-rich; TBS, tris-buffered saline; TLRs, toll-like receptors; TNF, tumor necrosis factor.CD5L (CD5 molecule-like) is a secreted glycoprotein that participates in host response to bacterial infection. CD5L influences the monocyte inflammatory response to the bacterial surface molecules lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by inhibiting TNF secretion. Here we studied the intracellular events that lead to macrophage TNF inhibition by human CD5L. To accomplish this goal, we performed functional analyses with human monocytic THP1 macrophages, as well as with peripheral blood monocytes. Inhibition of phosphatidylinositol 3-kinase (PtdIns3K) reversed the inhibitory effect of CD5L on TNF secretion. Among the various PtdIns3K isoforms, our results indicated that CD5L activates PtdIns3K (whose catalytic subunit is termed PIK3C3), a key modulator involved in autophagy. Further analysis revealed a concomitant enhancement of autophagy markers such as cellular LC3-II content, increased LC3 puncta, as well as LC3-LysoTracker Red colocalization. Moreover, electron microscopy showed an increased presence of cytosolic autophagosomes in THP1 macrophages overexpressing CD5L. Besides preventing TNF secretion, CD5L also inhibited IL1B and enhanced IL10 secretion. This macrophage anti-inflammatory pattern of CD5L was reverted upon silencing of autophagy protein ATG7 by siRNA transfection. Additional siRNA experiments in THP1 macrophages indicated that the induction of autophagy mechanisms by CD5L was achieved through cell-surface scavenger receptor CD36, a multiligand receptor expressed in a wide variety of cell types. Our data represent the first evidence that CD36 is involved in autophagy and point to a significant contribution of the CD5L-CD36 axis to the induction of macrophage autophagy.

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Section: Western Blot Analysis Of Cell Lysatesmentioning

confidence: 99%

The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

et al. 2015

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“…Cells were examined using the Nikon Eclipse Ti-E confocal microscope and the EZ-C1 and NIS-Element AR 3.00, sp7 software. The MAP-2 area and ratios of pCREB and pc-Jun over DAPI were quantified using ImageJ as described previously (23). The average size of A␤ aggregates was determined using the "analyze particles" function of ImageJ and the scale bar as a size reference (only aggregates Ͼ2 m 2 were analyzed).…”

Section: ϫ⌬Ctmentioning

confidence: 99%

C1q-induced LRP1B and GPR6 Proteins Expressed Early in Alzheimer Disease Mouse Models, Are Essential for the C1q-mediated Protection against Amyloid-β Neurotoxicity

Benoit

Hernandez

Dinh

et al. 2013

Journal of Biological Chemistry

131

120

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“…Nuclear factor of activated T cells (NFAT) translocation was also visualized using immunofluorescence as previously described 14 (online-only Data Supplement).…”

Section: Immunofluorescencementioning

confidence: 99%

Ginseng Reverses Established Cardiomyocyte Hypertrophy and Postmyocardial Infarction-Induced Hypertrophy and Heart Failure

Moey

Gan

Huang

et al. 2012

Circ: Heart Failure

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Background-A major challenge in the treatment of heart failure is the ability to reverse already-established myocardial remodeling and ventricular dysfunction, with few available pharmacological agents prescribed for the management of heart failure having demonstrated successful reversal of the remodeling and hypertrophic processes. North American ginseng (Panax quinquefolius) has previously been shown to effectively prevent cardiomyocyte hypertrophy and heart failure. Here, we determined whether North American ginseng can reverse established cardiomyocyte hypertrophy in cultured myocytes as well as hypertrophy and left ventricular dysfunction in experimental heart failure secondary to coronary artery occlusion. Methods and Results-Ginseng was administered in drinking water (0.9 g/L) ad libitum to rats after 4 weeks of sustained coronary artery ligation when heart failure was established or to angiotensin II-(100 nmol/L), endothelin-1-(10 nmol/L), or phenylephrine-(10 µmol/L) induced hypertrophic cultured neonatal ventricular cardiomyocytes. Echocardiographic and catheter-based measurements of hemodynamic parameters 4 weeks after starting ginseng treatment (8 weeks postinfarction) revealed nearly complete reversibility of systolic and diastolic abnormalities. Similarly, ginseng administration to hypertrophic cardiomyocytes resulted in a complete reversal to a normal phenotype after 24 hours as determined by cell surface area and expression of molecular markers. The effects of ginseng both in vivo and in cultured cardiomyocytes were associated with reversal of calcineurin activation and reduced nuclear translocation of the transcription factor NFAT3 (nuclear factor of activated T cells 3) in cultured myocytes. Moreover, the beneficial effect of ginseng was associated with normalization in the gene expression of profibrotic markers, including collagen (I and III) and fibronectin. Conclusions-This study demonstrates a marked ability of ginseng to reverse cardiac hypertrophy, myocardial remodeling, and heart failure, which was associated with and likely mediated by reversal of calcineurin activation. Ginseng may offer a potentially effective approach to reverse the myocardial remodeling and heart failure processes, particularly in combination with other treatment modalities.

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Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages

Cited by 158 publications

References 6 publications

The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

C1q-induced LRP1B and GPR6 Proteins Expressed Early in Alzheimer Disease Mouse Models, Are Essential for the C1q-mediated Protection against Amyloid-β Neurotoxicity

Ginseng Reverses Established Cardiomyocyte Hypertrophy and Postmyocardial Infarction-Induced Hypertrophy and Heart Failure

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