and mean percentage of YFP-positive cells, or and mean YFP intensity 24 h after treatment with 1 m Tg or 0.1% DMSO control. active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Furthermore, inhibition of IRE1 kinase activity or of downstream JNK USL311 activity prevented an increase in IRE1 levels during ER stress, suggesting that transcription is usually regulated through a positive feed-forward loop. Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent off-switch. and and Fig. S1, and and ATF6 protein levels were analyzed by Western blotting using ATF6 antibody. Actin served as loading control. real-time qPCR analysis of ATF6 USL311 mRNA in SH-SY5Y cells transduced with vectors encoding shRNA against ATF6 or control and treated with 3 m Tm or DMSO for 24 h. Results were normalized to -actin levels and expressed relative to DMSO-treated scram control cells (mean of = 3, indicate S.E. Student’s assessments were performed to compare Tm-treated and control group (* indicates < 0.05) or ATF6-KD and scram control cells (# indicates FAM194B < 0.05). 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. ATF6-reporter cells were transduced with shRNA against ATF6 or scrambled control vector. 96 h after transduction, cells were stained with Hoechst and PI. Images were taken at 1-h intervals starting immediately after treatment for 48 h using high-content time-lapse live cell imaging. indicate S.E. of all cells per time point and treatment. Data shown are representative of two experiments. YFP mean fluorescence intensity 30 h after treatment with 1 m Tg or 0.1% DMSO. indicate S.E. of = 3 wells ATF6-KD or = 2 wells scram. Student's assessments were performed comparing KD and scrambled groups. * USL311 indicates < 0.05. and and Fig. S2and schematic indicating reporter cell line and silencing construct used. and percentage of YFP-positive cells, or and mean YFP intensity over time in response to 1 1 m Tg or 0.1% DMSO in reporter cells transduced with silencing construct or scrambled control group was plotted. indicate S.E. of at least = 2 wells of a representative experiment. and mean percentage of YFP-positive cells, or and mean YFP intensity 24 h after treatment with 1 m Tg or 0.1% DMSO control. indicate S.E. of = 3 impartial experiments. Student's assessments were performed comparing KD and scrambled groups. * indicates < 0.05. and and Fig. S2, and and and Fig. S2, and and percentage of YFP-positive cells over time in response to 0.3 m Tm or 0.1% DMSO in ATF6-KD and scrambled control group was plotted. indicate S.E. of = 2 wells (ATF6-KD) or = 3 wells (mean percentage of YFP-positive cells 15 h after treatment. indicate S.E. of = 3 impartial experiments. Student's assessments were performed comparing ATF6-KD and scrambled control groups for each treatment. * indicates < 0.05. percentage of YFP-positive cells over time in response to 0.5 g/ml BFA or 0.1% DMSO in ATF6-KD and scrambled control group was plotted. indicate S.E. of = 3 wells (DMSO) or = 6 wells (mean percentage of YFP-positive cells 15 h after treatment. indicate S.E. of = 3 impartial experiments. Student's assessments were performed comparing ATF6-KD and scrambled control groups for each treatment. * indicates < 0.05. percentage of PI-positive cells over time in response to 0.3 m Tm or 0.1% DMSO in ATF6-KD and scrambled control group was plotted. indicate S.E. of = 3 wells (ATF6-KD) or = 2 wells (scram). mean percentage of PI-positive cells 45 h after treatment. indicate S.E. of = 3 impartial experiments. Student's assessments were performed comparing ATF6-KD and scrambled control groups for each treatment. percentage of PI-positive cells over time in response to 0.5 g/ml BFA or 0.1% DMSO in ATF6-KD and.
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