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. 2021 Jan 19;28(6):1941–1954. doi: 10.1038/s41418-020-00725-4

Fig. 5. PIWIL2 inhibits mTORC1 phosphorylation to regulate autophagy through IKKβ.

Fig. 5

a–c PIWIL2 overexpressed KYSE150 cells were treated with 10 μM IKK inhibitor BAY11-7082 for 12 h or co-transfected with p65 shRNA. a Autophagic flux were measured using RFP-GFP-LC3 method. b, c Protein level of autophagic markers (Beclin-1, p62, and LC3-II) were examined using WB. d KYSE150 cells were transfected with PIWIL2 expressed plasmid or empty vector. Phosphorylation level of mTORC1 and ULK1 were examined as markers of mTOR pathway. e Phosphorylation level of mTORC1 downstream targets (p85S6, p70S6, and 4E-BP1) were analyzed using WB in PIWIL2 overexpressed KYSE150 cells. f Autophage suppressed by PIWIL2 knockdown can be rescued by treatment of 100 nM rapamycin for 12 h to suppress mTORC1 activity in ESCC cell line KYSE150, KYSE510, and KYSE180. g Phosphorylation level of mTORC1 decreased by PIWIL2 overexpression can be rescued by BAY11-7082 treatment. h KYSE150 cells were transfected with different amount of PIWIL2 vector (1–3 µg). Immunoprecipitation of IKKβ by anti-PIWIL2 or anti-TSC1 was performed to determine the competitiveness of interaction. i Cell-free expression of PIWIL2, IKKβ, and TSC1 were conducted using the TnT System, followed with immunoprecipitation analysis. j The mTORC1 pathway suppressed by PIWIL2 overexpression can be rescued by co-transfection with Rheb. k IKKβ K44A (Kinase-dead variant) was constructed and transfected into KYSE150 cells to rescue knockdown of IKKβ by transfecting shRNA against IKKβ (sh-IKKβ). *p < 0.05; **p < 0.01.