S6K1 knockdown and inhibition reduce necroptosis in HT22 cells. (IMAGE)
Caption
(A, G) Western blotting showed increased levels of p-S6K1/S6K1, p-S6/S6, p-MLKL/MLKL, and p-RIP3/RIP3 expression following TSZ application. These increases were attenuated by pretreatment with S6K1 siRNA and an S6K1 inhibitor before TSZ application. (B–E, H–K) Statistical results of Western blotting. (F) Enhanced immunofluorescence intensity of p-S6K1 in HT22 cells after TSZ application. (L) Phase-contrast images showed changes in necrotic cells following TSZ application, as well as after pretreatment with S6K1 siRNA and inhibitor before TSZ application. Microscopic magnification, 20 ×. (M) PI staining (red) indicated the decreased changes in necrotic cells after pretreatment with S6K1 siRNA and inhibitor before TSZ application compared with TSZ application. Nuclei were stained with DAPI (blue). (N) LDH assay indicated the decreased percentage of cell death after pretreatment with S6K1 siRNA and inhibitor before TSZ application compared with TSZ application. (O) Statistical results of the PI-positive necrotic cells. n = 3, 4, 5, 6, or 8. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 versus the CTL or TSZ group. Scale bar: 50 μm.
Credit
Shuchao Wang, Yating Tan, Minghai Hu, Meijuan Wang, Lu Liang, Xing Luo, Dan Chen, Bing Jiang, Ceshi Chen, Jufang Huang, Kun Xiong
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