Fig 1: Necroptosis induced by TNF destabilizes SNARE complexes and cleaves STX17 to block LC3 degradation. (A) L929 cells stably expressing GFP-SNAP29 were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. (B) L929 cells stably expressing GFP-SNAP29 were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ), TQ plus 5 µM GSK’872 (TQG), or TQ plus 5 µM necrostatin-1 (TQN) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. (C) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17 and ACTB. (D) L929 WT, ripk3 KO or mlkl KO cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17, RIPK3, MLKL, and GAPDH. (E) L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (siPrkaa1/siPrkaa2). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) with or without 5 µM GSK’872 (G) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (siCtrl, medium). Statistical graphics represents mean + SD (n = 3). Statistical analysis was done by ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). Statistically significant differences are only indicated for TQ and TQG (scr vs. Prkaa1/Prkaa2 siRNA) and for TQ vs. TQG (within each treatment). Statistically significant differences to control (siCtrl, medium) are depicted as letters directly above the bars. ** or b: P < 0.01, *** or c: P < 0.001, ns: non-significant. (F) L929 cells were left untransfected or transiently transfected with cDNA encoding 3xFLAG-MmRIPK3 for 24 h. Then untransfected cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 3 h. Cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins
Fig 2: Gene expression in INS(832/13) cells. mRNA expression in cells cultured in LG, NT or EBSS in the presence or absence of E16 or UV-inactivated E16 (E16UV): Atg3 (a), Atg5 (b), Atg7 (c), Atg9a (d), Atg10 (e), Atg12 (f), Lamp2 (g), Stx17 (h) and Uvrag (i). Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001
Fig 3: LncRNA-GAS5 blocks STX17 expression by preventing YY1 from recruiting STX17 promoter in Acinetobacter baumannii infection. 6A: HeLa cells were transfected with pc3.1-YY1 plasmid into HeLa cells for different times. The level of YY1 was assessed by Western blot assay. 6B: HeLa cells were transfected with sh-NC or sh-YY1 plasmid (sh-YY1-si1, sh-YY1-si2, sh-YY1-si3) into HeLa cells for 48 hours. The level of YY1 was assessed by Western blot assay. 6C: The expression level of autophagy related proteins in HeLa cells transfected with pcDNA3.1 or pc3.1-YY1 plasmid were detected by Western blot assay. 6D: The expression level of autophagy related proteins in HeLa cells transfected with sh-NC or sh-YY1 plasmid were detected by Western blot assay. 6E: Dual-luciferase reporter assays of STX17 promoter activity in HeLa cells which were transfected with pcDNA3.1 or pc3.1-YY1 plasmids respectively. 6F: Dual-luciferase reporter assays of STX17 promoter activity in HeLa cells which were transfected with sh-NC or sh-GAS5 plasmids respectively. 6G: HeLa cells were transfected with pcDNA3.1, pc3.1-GAS5 or co-transfection of pc3.1-GAS5 and sh-YY1 respectively, and then STX17 expression was detected by Western blot assay. 6H: HeLa cells were transfected with sh-NC, sh-GAS5 or co-transfection of sh-GAS5 and pc3.1-YY1 respectively, and then STX17 expression was detected by Western blot assay.6I: After transfected with pcDNA3.1, pc3.1-GAS5 or co-transfection of pc3.1-GAS5 and sh-YY1 respectively, the activity of STX17 promoter in HeLa cells were detected by Dual-luciferase reporter assay. 6J: After transfected with sh-NC, sh-GAS5 or co-transfection of sh-GAS5 and pc3.1-YY1, the activity of STX17 promoter in HeLa cells were detected by Dual-luciferase reporter assay. 6K: The expression of YY1 was evaluated by Western blot assay after transfected with pcDNA3.1 or pc3.1-GAS5 as well as sh-NC or sh-GAS5 plasmid respectively. 6L: The enrichment of YY1 at STX17 promoter region was evaluated via ChIP assay in HeLa cells transfected with pc3.1-GAS5 or pcDNA3.1. 6M: The expression of YY1 in HeLa cells infected with Acinetobacter baumannii for different times was detected by Western blot assay. 6N-O: Western blotting was used to detect the expression of STX17 in HeLa cells which the pc3.1-GAS5 and sh-YY1 plasmids or sh-GAS5 and YY1 plasmids co-transfected for 24 hours, and then infected with Acinetobacter baumannii for 24 hours
Fig 4: ULK1-2A limits ULK1–STX17 interaction, resulting in defective macroautophagy.A and B, ULK1-2A decreased interaction with STX17. Cells were transfected with FLAG-ULK1-WT or FLAG-ULK1-2A and then treated with CQ. Lysates were subjected to immunoprecipitation and immunoblotting as indicated. Quantitation indicates the ratio of the FLAG signal normalized to STX17 in the IP (A) or the ratio of the STX17 signal normalized to FLAG in the IP (B). ∗∗∗ indicates p < 0.001. C, cells were transfected with FLAG-ULK1-WT or FLAG-ULK1-2A and treated with CQ. Autophagic levels were monitored with LC3B antibodies. D, cells were transfected with FLAG-ULK1-WT or FLAG-ULK1-2A plasmids and then stained with anti-LC3B antibodies and DAPI. The scale bar represents 10 μm. E, quantitation of LC3B-positive cells. WT cells were normalized as 1. ∗ indicates significant differences as determined by Student’s t test (p < 0.05). CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; IP, immunoprecipitation; ULK1, Unc-51-like kinase 1.
Fig 5: Kansl1+/- mice exhibit autophagic defects and impaired neuronal and cardiac functions.a, c Representative immunofluorescence images of hippocampus CA1 regions (a) and hearts (c) with LC3B antibody (red). Nuclei are labeled with DAPI (blue). Scale bar, 10 µm. b, d Quantification of LC3B puncta intensity in (a) and (c). LC3B puncta intensity in Kansl1+/+ was normalized to ‘1’ (n = 3 mice in each group). e Electron microscopy images of hippocampus CA1 regions and hearts. N, nucleus. Arrows indicate autophagosomes. Scale bar, 500 nm. f Violin plot showing the number of autophagosomes per µm2 in (e). n = 22, 14, 18, 21 cells from 3 independent experiments. g Representative immunofluorescence images of hippocampus CA1 regions and hearts with Stx17 antibody (green). Nuclei are labeled with DAPI (blue). Scale bar, 10 µm. h Quantification of Stx17 intensity in (g). Stx17 intensity in Kansl1+/+ was normalized to ‘1’ (n = 6, 8, 3, 3 mice from left to right). i Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object versus both the novel object and the familiar object in the choice trial. n = (10 Kansl1+/+ mice and 10 Kansl1+/- mice). j Morris water maze test. Mean escape latencies time to find the platform. n = 10 Kansl1+/+ mice and 10 Kansl1+/- mice. k Representative images of Nissl staining for whole brain of Kansl1+/+ and Kansl1+/- mice. Scale bar, 500 µm. l Representative images of dendritic spine of hippocampus CA1 regions visualized with Golgi-cox staining. Scale bar, 5 µm. m Quantification of spine density in (l). n = 29, 21 (from 3 Kansl1+/+ and 2 Kansl1+/- mice). n Representative images of echocardiograms on mice. Scale bar, 100 ms. o Echocardiographic analyses of (n). Left, ejection fraction. Right, fractional shortening. n = (24 Kansl1+/+ mice and 21 Kansl1+/- mice). Mice tested above were about 5 months old. Source data are provided as a Source data file. All data are means ± SEM. b, d, f, h, i, m, o Two-tailed Student’s t-tests; j by two-way ANOVA test with Dunnett’s multiple hoc test.
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