a.u., arbitrary units.Įmploying high-content live cell imaging to interrogate cross-talk between the UPR-signaling branches. Student's t tests were performed comparing KD and scrambled groups. of n = 3 wells ATF6-KD or n = 2 wells scram. D, YFP mean fluorescence intensity 30 h after treatment with 1 μ m Tg or 0.1% DMSO. Data shown are representative of two experiments. of all cells per time point and treatment. Images were taken at 1-h intervals starting immediately after treatment for 48 h using high-content time-lapse live cell imaging. 96 h after transduction, cells were stained with Hoechst and PI. ATF6-reporter cells were transduced with shRNA against ATF6 or scrambled control vector. C, YFP mean fluorescence intensity over time in ATF6-reporter cells transduced with ATF6-KD or scram control construct, treated with Tg or 0.1% DMSO, respectively. Student's t tests were performed to compare Tm-treated and control group (* indicates p < 0.05) or ATF6-KD and scram control cells (# indicates p < 0.05). Results were normalized to β-actin levels and expressed relative to DMSO-treated scram control cells (mean of n = 3, error bars indicate S.E. B, real-time qPCR analysis of ATF6 mRNA in SH-SY5Y cells transduced with vectors encoding shRNA against ATF6 or control and treated with 3 μ m Tm or DMSO for 24 h. A, ATF6 protein levels were analyzed by Western blotting using ATF6 antibody. SH-SY5Y cells were transduced with vectors encoding shRNA against ATF6 or scrambled control ( scram). Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent "off-switch."ĪPY29 ATF6 GRP78 IRE1 UPR reporters X-box binding protein 1 (XBP1) c-Jun N-terminal kinase (JNK) cell death endoplasmic reticulum stress (ER stress) fluorescent reporter high content imaging imaging lentivirus protein misfolding stress response unfolded protein response (UPR). Furthermore, inhibition of IRE1 kinase activity or of downstream JNK activity prevented an increase in IRE1 levels during ER stress, suggesting that IRE1 transcription is regulated through a positive feed-forward loop. Moreover, overexpression of the transcriptionally active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Transient increases in both IRE1 mRNA and IRE1 protein levels were observed in response to ER stress, suggesting that IRE1 up-regulation is a general feature of ER stress signaling and was further increased in cells lacking ATF6 expression. We observed that loss of ATF6 expression results in uncontrolled IRE1-reporter activity and increases X box-binding protein 1 ( XBP1) splicing. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR-reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signaling proteins were silenced through lentivirus-delivered shRNA constructs.
When this restoration fails, however, cells undergo apoptosis. These proteins initiate a signaling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated.
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