p53

A, HAECs were per-incubated with PFTα (3 μmol/L) for 60 min followed by transfected with siB-myb or siNC in the presence of PFTα (3 μmol/L) for 7 d. The expression levels of B-myb, p-p53, p53 and p21 proteins were analysed by western blotting. GAPDH was used as a loading control. A typical group of blots is shown from one of three independent experiments.

Nutlin-3a preserved p53 expression without influencing high glucose (HG)-induced podocyte impairment. A-D: cultured podocytes were pre-treated by nutlin-3a for 2 hrs before subjected to HG treatment. Western blotting gel documents (A) and summarized data (B) showing the expression of p53 and MDM2 in podocytes under HG exposure for 24 hrs. n = 4. Western blotting gel documents (C) and summarized data (D) showing the expression of Desmin in podocytes under HG exposure for 24 hrs. n = 3. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Vehl + HG. Ctrl: control; Vehl: vehicle; nutlin-3a: nutlin-3a treatment.

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells

A, HAECs were per-incubated with PFTα (3 μmol/L) for 60 min followed by transfected with siB-myb or siNC in the presence of PFTα (3 μmol/L) for 7 d. The expression levels of B-myb, p-p53, p53 and p21 proteins were analysed by western blotting. GAPDH was used as a loading control. A typical group of blots is shown from one of three independent experiments.

Nutlin-3a preserved p53 expression without influencing high glucose (HG)-induced podocyte impairment. A-D: cultured podocytes were pre-treated by nutlin-3a for 2 hrs before subjected to HG treatment. Western blotting gel documents (A) and summarized data (B) showing the expression of p53 and MDM2 in podocytes under HG exposure for 24 hrs. n = 4. Western blotting gel documents (C) and summarized data (D) showing the expression of Desmin in podocytes under HG exposure for 24 hrs. n = 3. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Vehl + HG. Ctrl: control; Vehl: vehicle; nutlin-3a: nutlin-3a treatment.

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells