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. 2018 Nov 14;9(11):1137. doi: 10.1038/s41419-018-1172-y

Fig. 5. Glucocorticoid promoted microtubule destabilization and following transport impairment.

Fig. 5

a The cells were treated with cortisol (1 μM) for 24 h and lysed. α-tubulin was co-immunoprecipitated with an anti-SCG10 antibody (the left side). Expression of SCG10 and α-tubulin in total cell lysates is shown in the right side. ** indicates p < 0.01 vs. control. n = 4. b The cells were pretreated with rapamycin (100 nM) for 30 min before cortisol (1 μM) for 48 h. Acetylated α-tubulin, tyrosinated α-tubulin, and α-tubulin were detected with western blot. ** indicates p < 0.01 vs. control and ## indicates p < 0.01 vs. cortisol alone. n = 4. c The cells were treated with cortisol (1 μM) for 24 h. α-tubulin was co-immunoprecipitated with an anti-kinesin-1 antibody (the left side). Expression of kinesin-1 and α-tubulin in total cell lysates is shown in the right side. ** indicates p < 0.01 vs. control. n = 4. d The cells were incubated with cortisol (1 μM) for 24 h. Co-localization of α-tubulin (red) and kinesin-1 (green) was visualized with SRRF imaging system. DAPI was used for nuclear counterstaining (blue). ** indicates p < 0.01 vs. control. Scale bars represent 20 μm (magnification, ×1000). n = 5. e The hippocampus of mice exposed to rapamycin (8 mg/kg) for 2 days before corticosterone (10 mg/kg) for 24 h was collected. Acetylated α-tubulin, tyrosinated α-tubulin, and α-tubulin were detected by western blot. ** indicates p < 0.01 vs. vehicle. ## indicates p < 0.01 vs. corticosterone alone. n = 5. f The hippocampus of mice exposed to rapamycin (8 mg/kg) for 2 days before corticosterone (10 mg/kg) for 24 h was collected and lysed. α-tubulin was co-immunoprecipitated with an anti-kinesin-1 antibody (the left side). Expression of kinesin-1 and α-tubulin in total cell lysates is shown in the right side. ** indicates p < 0.01 vs. vehicle. # indicates p < 0.05 vs. corticosterone alone. n = 4. g–h The cells were incubated with cortisol (1 μM) for 48 h. Co-localization of α-tubulin (red) and AMPAR1/2 (green) was visualized with SRRF imaging system. DAPI was used for nuclear counterstaining (blue). *, ** indicates p < 0.05, p < 0.01 vs. control, respectively. Scale bars represent 20 μm (magnification, ×1000). n = 5. i The cells treated with cortisol (1 μM) for 48 h were immunostained with DAPI (blue) and COX IV (green). ** indicates p < 0.01 vs. control. Scale bars represent 20 μm (magnification, ×1000). n = 5. j The cells were pre-incubated with paclitaxel (10 μM) for 30 min before cortisol (1 μM) for 48 h. After treatment, water soluble tetrazolium salt (WST-1) assay was performed to measure cell viability. ** indicates p < 0.01 vs. control and # indicates p < 0.05 vs. cortisol alone. n = 6. All blot and immunofluorescence images are representative. Quantative data are presented as a mean ± S.E.M.