Journal: Oncogene

Article Title: VPS34 stimulation of p62 phosphorylation for cancer progression

doi: 10.1038/onc.2017.295

Figure Lengend Snippet: Inhibition of caspase 8 promotes the fusion of autophagosome with lysosome. ( a ) Inhibition of LBH589-induced apoptosis by caspase inhibitors. Apoptotic cell death of MCF-7 cells induced by treatment with the indicated concentrations of LBH589 and/or caspase inhibitors for 48 h. For caspase inhibitor treatment, cells were pretreated with 10 μ M z-VAD-FMK (Z-VAD) or z-IETD-FMK (IETD) for 4 h. ( b ) Z-VAD or IETD inhibited LBH589-induced cleavage of caspase 7, caspase 8, caspase 9 and PARP-1 in MCF-7 cells treated as indicated for 24 h. ( c ) Cell viability of MCF-7 cells treated as indicated for 24 h. ( d ) Accumulation of Vps34, LC3 and p62 in MCF-7 cells treated as indicated with or without 20 μM chloroquine for 24 h. ( e ) Confocal microscopic evaluation of autophagosome-lysosome fusion in MCF-7 cells. MCF-7 cells stably expressing EGFP-LC3 (Green) treated as indicated for 18 h were stained with lysosome tracker staining (Red). Immunofluorescence analyses were performed using confocal microscopic detection (63 × oil). Left, representative confocal images. Right, relative Green + red + dots per cell calculated from 20 cells; bars, s.d. ** P <0.01. ( f ) Disruption of autophagosome-lysosome fusion in MCF-7 cells. MCF-7 cells stably expressing tfLC3 were treated as indicated for 18 h. Immunofluorescence analyses were performed using confocal microscopic detection (60 × oil). Left, representative confocal images. Right, % of autolysosomes per cell was calculated from 20 cells by using the formula of (1-yellow dots/red dots) × 100; bars, s.d. ** P <0.01. DAPI, 4', 6-diamidino-2-phenylindole.

Article Snippet: The vector encoding mRFP-GFP-LC3 (tfLC3) was excised from ptfLC3 (Addgene, 21074) and subcloned into the lentiviral vector pCDH1-MCS1-EF1-puro (System Biosciences, CD510A-1).

Techniques: Inhibition, Stable Transfection, Expressing, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: The role of microglial LRRK2 in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction

doi: 10.1101/2023.04.03.535418

Figure Lengend Snippet: (A) After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), striatum and midbrain tissues were analyzed for the protein levels of NLRP3, cleaved CASP1, active CTSB, and LAMP1 by western blotting. (B) BV2 cells were examined for protein-protein interactions of LRRK2 with RAB10, fluorescence intensity, and colocalization of NLRP3 and LRRK2, RAB10 in the microglia. (C) Following Mn exposure for 12 and 24 h, LRRK2 WT and G2019S BV2 cells were assessed for autophagic flux with LC3-mcherry-GFP fluorescence assay. Cells with high autophagic flux were determined and quantified by red fluorescence using flow cytometry. (D) Following pre-treatment of LRRK2 inhibitor MLi-2 (50 nM, 0.5 h) and Mn exposure (250 µM, 12 h), LRRK2 WT and G2019S-expressing BV2 cells were analyzed for lysosomal activity by lysotracker assays. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, compared with the controls; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, @@@@ p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3-5). Data are expressed as mean ± SD.

Article Snippet: Expression vectors for 2XMyc-LRRK2-WT (25361), 2XMyc-LRRK2-G2019S (25362), pDEST53-LRRK2-WT (25044), pDEST-LRRK2-G2019S (25045), EGFP-RAB10 (49472), EGFP-RAB10T23N (49545), and 2xmyc-RAB10 (164631) and reporter LC3-mCherry-GFP vector (110060) were from Addgene (Watertown, MA).

Techniques: Western Blot, Fluorescence, Flow Cytometry, Expressing, Activity Assay

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a KEGG pathway enrichment of DEGs in ZIKV-infected U251 cells transfected with HA-DISC1 (1 μg) plasmid compared with the control group transfected with pcDNA. b KEGG pathway enrichment of differentially expressed proteins identified by IP/MS analysis in U251 cells overexpressing HA-DISC1 (5 μg) plasmid compared with the control group transfected with pcDNA. c Chord diagram illustrates the proteins identified in the IP/MS analysis. d U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h followed by ZIKV infection. The protein levels of AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot at 0, 12, 24, and 36 h post-infection. e U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, rapamycin (25 μM), and MHY1485 (10 μM) for 24 h. ZIKV E expression was assessed by Western blot. f U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, autophagosome inhibitor 3-MA (1 mg/ml), and lysosome inhibitor CQ (100 μM) for 24 h. ZIKV E expression was assessed by Western blot. KEGG pathway enrichment analyses in ( a , b ) were performed using a one-tailed Fisher’s exact test. Images in ( d − f ) are one representative experiment out of three independent replicates with similar results ( n = 3). Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Infection, Transfection, Plasmid Preparation, Control, Protein-Protein interactions, Western Blot, Expressing, One-tailed Test

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Conserved LIR motifs (amino acids 210 FSFI 213) in DISC1 were highlighted in yellow, which were mutated into AAAA in DISC1 mutant. b 293T cells were co-transfected with HA-DISC1 (5 μg) or HA-DISC1 mutant (5 μg) and GFP-LC3 (5 μg) plasmids for 48 h. Cellular lysates were subjected to immunoprecipitation with anti-Flag or anti-HA magnetic beads and Western blot assays using the indicated antibodies. c − e U251 cells were transfected with HA-DISC1 (1 μg) or HA-DISC1 mutant (1 μg) plasmids for 24 h, followed by ZIKV infection for 24 and 48 h. Viral mRNA levels, protein expression and titers were measured on D1 and D2 post-infection by qRT-PCR ( c ), Western blot ( d ), and plaque assays ( e ). Images in ( b , d ) are from one representative experiment out of three independent replicates with similar results ( n = 3). Data shown in ( c , e ) are from one representative experiment out of three independent replicates with similar results, each including 3 biological replicates per group ( n = 3). Data shown in ( c , e ) were confirmed to follow a normal distribution using the Shapiro-Wilk test, and statistical analysis was performed using two-tailed one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Infection, Expressing, Quantitative RT-PCR, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Primary cortical neurons were isolated from neonatal mice (P0–P1) and induced to differentiate in vitro. On day 7 of differentiation, neurons exhibiting neurite outgrowth were observed under bright-field microscopy. Confocal imaging showed neuronal nuclei stained with DAPI (blue), and endogenous Neun labeled with an anti-Neun antibody (red). Scale bar = 50 μm. b , c WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. Viral mRNA levels and titers were measured on D1 and D2 post-infection using qRT-PCR ( b ) and plaque assay ( c ). d WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. The protein levels of ZIKV E protein, DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot on D1 and D2 post-infection. e, f The ratios of pAMPKα to AMPKα ( e ) and pmTOR to mTOR ( f ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8d. g − i Quantification of P62 ( g ), LC3A/BⅠ ( h ) and LC3A/BⅡ ( i ) protein levels relative to GAPDH at each time point in Fig. 8d. j , k Placentas ( j ) and fetal heads ( k ) from non-infected and ZIKV-infected WT and Disc1 KD mice were collected on E13.5. The protein levels of DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot. l − o The ratios of pAMPKα to AMPKα ( l, m ) and pmTOR to mTOR ( n , o ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8j ( l, n ) and Fig. 8k ( m , o ). p − s Quantification of P62 ( p , q ) and LC3A/BⅡ ( r , s ) protein levels relative to GAPDH at each time point in Fig. 8j ( p , r ) and Fig. 8k ( q , s ). Data shown in ( b , c ) are from one representative experiment out of three independent replicates with similar results, each including 3 biological replicates per group ( n = 3). Images in ( a , d , j – k ) are from one representative experiment out of three independent replicates with similar results ( n = 3). Data shown in ( e − i , l − s) are collected from three independent experiments ( n = 3). Data shown in ( b − c , e − i , l − s ) were confirmed to follow a normal distribution using the Shapiro-Wilk test, and statistical analysis was performed using Student’s unpaired two-tailed t test ( b , c ) or two-tailed one-way ANOVA followed by Tukey’s multiple comparison test ( e − i , l − s ). Data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Isolation, In Vitro, Microscopy, Imaging, Staining, Labeling, Incubation, Infection, Quantitative RT-PCR, Plaque Assay, Western Blot, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a 6-8-weeks-old WT or Disc1 KD mice were treated with 2 mg MAR1-5A3 on the day prior to infection and then intraperitoneally (i.p.) inoculated with PBS or 1 × 10 6 PFU of ZIKV. Mouse brains were collected on D6 post-infection. Representative H&E-stained sections of the hippocampal DG and CA3 regions are shown. Black arrows indicate shrunken, hyperchromatic neurons with indistinct boundaries Scale bar = 100 μm. b Representative immunofluorescence-stained sections of the hippocampal DG region. Cell nuclei were stained using DAPI (blue). Endogenous Neun was labeled with anti-Neun antibody (red). Endogenous LC3A/B was labeled with anti-LC3A/B antibody (yellow). Scale bar = 100 μm. Representative images in ( a , b ) are from one independent experiment, including 6 mice per group ( n = 6), consisting of 3 males and 3 females.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Infection, Staining, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Neonatal mice were intracranially injected with 200 PFU of ZIKV GZ01 or an equal volume of PBS into the λ point of the brain on postnatal day 2 (P2). Mouse brains were collected on D6 post-infection. Representative H&E-stained sections of the hippocampal CA1 region are shown. Black arrows indicate shrunken, hyperchromatic neurons with indistinct boundaries. Orange arrows indicate nuclear pyknosis and fragmentation. Scale bar = 100 μm. b Representative immunofluorescence-stained sections of the hippocampal DG region. Cell nuclei were stained using DAPI (blue). Endogenous Neun was labeled with anti-Neun antibody (red). ZIKV E protein was labeled with anti-E antibody (green). Endogenous LC3A/B was labeled with anti-LC3A/B antibody (yellow). Scale bar = 100 μm. Representative images in ( a , b ) are from one representative experiment out of two independent experiments with similar results, each including 4 mice per group ( n = 4), consisting of 2 males and 2 females.

Article Snippet: The GFP-tagged LC3 expression plasmid was constructed by inserting the GFP-tagged LC3 coding sequence (NCBI accession number: NM_022818.5 ) into the pcDNA vector in Sangon Biotechnology (Shanghai, China).

Techniques: Injection, Infection, Staining, Immunofluorescence, Labeling