Extended Data Fig. 3: Nuclear IDO1 is degraded upon low ROS.
From: IDO1 regulating ROS rhythm reveals glycogenolysis/PPP as a cancer treatment target
(a) The intensity of HyPerRed fluorescence of synchronized MCF-7 and U2OS cells. (b)The relative mRNA level of IDO1 in MCF-7 and U2OS cells was analyzed by real time qPCR. (c, d) The expression of cytosolic or nuclear IDO1 in synchronized U2OS cells at different time points was analyzed by Western blot (c) and the normalized IDO1 intensity was calculated (d). (e-g) The IDO1 location of synchronized U2OS cells at different time points were observed by confocal microscope (e). Scale bar, 10 μm. The ratio of nuclear/cytosolic IDO1 fluorescence intensity (f) and the relative HyPerRed fluorescence intensity were calculated (g). (h) The expression of cytosolic or nuclear IDO1 in synchronized MCF-7 cells at different time points was analyzed by Western blot. (i) The location of IDO1 mRNA in MCF-7 and U2OS cells was analyzed by RNA-Scope and the relative IDO1 mRNA were calculated. (j) The expression of cytosolic or nuclear IDO1 in MCF-7 and U2OS cells treated with 100 ng/mL WGA or 20 μM Pitstop2 for 12 h was analyzed by Western blot. (k, l) The IDO1 location of MCF-7 and U2OS cells treated with 100 ng/mL WGA or 20 μM Pitstop2 for 12 h were observed by confocal microscope (k). Scale bar, 10 μm. The ratio of intensity of cytosolic and nuclear IDO1 was calculated (l). n = 6 technical replicates. (m) The intensity of HyPerRed fluorescence of Synchronized MCF-7 treated with 20 μM Pitstop2 for 12 h were detected by flow cytometry for 48 h. For a-j, m, n = 3 biological independent experiments. All error bars are mean ± SD. P values were calculated by two-tailed Student’s t test (i) and one-way ANOVA followed by Bonferroni’s test (l).
