Fig 1: TIPE2 inhibits autophagy levels in H9c2 cells. (A) Levels of autophagy were measured using a tandem sensor. OGD increased autophagy levels in H9c2 cells, while the overexpression of TIPE2 reduced autophagy levels in H9c2 cells with/without OGD. (B) Western blotting was used to analyze the proteins expression levels of autophagy-related LC3B and ATG14 in H9c2 cells. Data are representative of three independent repeats per experiment. &P>0.05 vs. plasmid; *P<0.05, ***P<0.001 vs. plasmid; #P<0.001 vs. plasmid + OGD. TIPE2, TNF-a-induced protein 8-like protein 2; OGD, oxygen glucose deprivation; over-, overexpression; ATG14, autophagy related 14.
Fig 2: Activation of autophagy by KA. Expression of LC3/Atg8, Atg5, Atg14, Beclin-1, and Lamp1 increased in primary neurons treated with KA (1 µM). The expression of p62 decreased after KA. Both changes are reversed by cocultured astrocytes, and this effect is differently abolished by AG1024, LY294022, and SB203580. ß-Actin was used as a loading control. *KA neurons vs. neurons, p < 0.05; #versus KA neurons, p < 0.05. n = 3 in each group, and the data was expressed as mean ± SD. WB: the left panel shared the same internal loading control, and the right panel also shared the same control.
Fig 3: CircMUC16-miR-199a-5p-Beclin1 axis regulated autophagy. a The expression of miRNAs of SKOV3 cells was determined by qPCR. b The expression of miRNAs of A2780 cells was determined by qPCR. c The location of miR-199a and circMUC16 in SKOV3 cells was detected using FISH assay. d RNA pulled down assay was performed. Then, the expression of circMUC16 was detected by qPCR. e RNA pulled down assay was performed. Then, the expression of miR-199a was detected by qPCR. f The expression of Beclin1 and ATG14 of SKOV3 cells was determined using western blot. f SKOV3 cells were transfected with LV2-NC, LV2–1, LV2–2, LV2–1 + miR-199a inhibitors, miR-199a mimics, miR-199a mimic inhibitors. Then, the expression of Beclin1 and ATG14 were detected using western blot. g SKOV3 cells were co-transfected with miR-199a mimic or control RNA (NC) using luciferase reporter plasmids containing either wild-type (pMIR-CircMUC16) or mutant (pMIR-CircMUC16m). Luciferase expression was measured. Error bars represent the standard error. The symbols * and ** indicate p < 0.05 and 0.01, respectively
Fig 4: TIPE2 reduces autophagy and OGD-induced apoptosis in H9c2 cells by activating the mTORC1 signaling pathway. (A) Western blotting was used to analyze the protein expression levels of TIPE2, Raptor, p-mTOR/mTOR and p-4EBP1/4EBP1 in H9c2 cells following different treatments. (B) Semi-quantification of the expression levels presented in part (A). (C) Expression levels of autophagy-related proteins, LC3B and ATG14, in H9c2 cells following different treatments. (D) TUNEL staining was used to detect OGD-induced apoptosis in H9c2 cells. Data are representative of three independent repeats per experiment. *P<0.05, **P<0.01, ***P<0.001 vs. plasmid; #P<0.05 and ###P<0.001 vs. over-TIPE2; &P<0.05 vs over-TIPE2 + ODG. TIPE2, TNF-a-induced protein 8-like protein 2; OGD, oxygen glucose deprivation; over-, overexpression; ATG14, autophagy related 14; p-, phosphorylated; Raptor, regulatory associated protein of mTOR complex 1; 4EBP1, eukaryotic translation initiation factor 4E.
Fig 5: Overexpressing ATG14 promoted CACO-2 autophagy in the presence of miR-148b-3p mimic through increasing ATG14. A, B Apigenin promoted the autophagy of CACO-2 after co-cultured with miR-148-3p mimic. **p < 0.01 vs miNC; ###p < 0.001 vs mimic. C Apigenin did not affect miR-148b-3p in CACO-2 cells after co-cultured with miR-148b-3p mimic. D Apigenin treatment enhanced the protein level of ATG14 in CACO-2 cells in the presence of miR-148-3p mimic
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