Erythropoietin (Epo) receptor (EpoR) is expressed in several cancer cell lines, and the functional consequence of this expression is under extensive study. In this study, we used a cervical cancer cell line in which EpoR was first found to be expressed and to correlate with the severity of the disease. We demonstrate that Epo is a chemoattractant for these cancer cells, enhancing their migration under serum-starved conditions. Using a Transwell migration system, we show that Epo enhances cancer cell migration in a dose-and time-dependent manner. The effect of Epo is dependent on the activity of two signaling pathways: the mitogen-activated protein kinase (MAPK) pathway and the RhoA GTPase pathway. We show that Epo activates both pathways in a Janus kinase-dependent manner and that this activation is required for Epo effects on cell migration. Furthermore, we use both pharmacological and genetic inhibitors to demonstrate that the activation of RhoA GTPase is dependent on the activity of the MAPK pathway, providing the first evidence for interaction between these two signaling cascades.Erythropoietin (Epo) is a cytokine that regulates red blood cell production through binding to its cell surface receptor expressed in erythroid progenitor cells (D'Andrea et al., 1989). Binding of Epo to its receptor induces dimerization of two receptor subunits and subsequent activation of the associated Janus kinase (Jak)2 (Witthuhn et al., 1993). This leads to phosphorylation of several tyrosine residues in Epo receptor (EpoR) and recruitment of SH2-containing proteins, which result in activation of several cascades of signal transduction pathways, notably the Ras/extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) (Torti et al., 1992) pathway and the phosphatidylinositol 3/Akt kinase pathway (Damen et al., 1995). Jak2 also phosphorylates signal transducer and activator of transcription (Stat)5 and Stat3 (Kirito et al., 1997), which then translocate to the nucleus to act as transcription factors and mediate Epo effects. In addition to Ras, Epo has been shown to activate other members of the Ras superfamily such as Rac
Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as "typical" or high density "hotspot" TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both "standard" and "high" settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R = 0.8972, hotspot: R = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R = 0.8036, standard/hotspot: R = 0.4309, high/typical: R = 0.7895, high/hotspot: R = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.
Objectives The chick is rapidly becoming a standardized preclinical model in vision research to study mechanisms of ocular disease. We seek to comprehensively evaluate the N-methyl-D-aspartate (NMDA) model of excitotoxic retinal damage using multimodal imaging, functional, and histologic approaches in NMDA-damaged, vehicle-treated, and undamaged chicks. Methods Chicks were either left undamaged in both eyes or were injected with NMDA in the left eye and saline (vehicle) in the right eye. TUNEL assay was performed on chicks to assess levels of retinal cell death one day post-injection of NMDA or saline and on age-matched untreated chicks. Spectral domain optical coherence tomography (SD-OCT) was performed weekly on chicks and age-matched controls day 1 (D1) up to D28 post-injection. Light adapted electroretinograms (ERG) were performed alongside SD-OCT measurements on post-injection chicks along with age-matched untreated controls. Results Untreated and vehicle-treated eyes had no TUNEL positive cells while NMDA-treated eyes accumulated large numbers of TUNEL positive cells in the Inner Nuclear Layer (INL), but not other layers, at D1 post injection. Significant inner retina swelling or edema was found on SD-OCT imaging at D1 post-injection which resolved at subsequent timepoints. Both the INL and the inner plexiform layer significantly thinned by one-week post-injection and did not recover for the duration of the measurements. On ERG, NMDA-treated eyes had significantly reduced amplitudes of all parameters at D1 with all metrics improving over time. The b-wave, oscillatory potentials, and ON/OFF bipolar responses were the most affected with at least 70% reduction immediately after damage compared to the fellow eye control. Conclusion This study establishes a normative baseline on the retinal health and gross functional ability as well as intraocular pressures of undamaged, vehicle-treated, and NMDA-damaged chicks to provide a standard for comparing therapeutic treatment studies in this important animal model.
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