Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

doi: 10.1038/labinvest.2009.8

Figure Lengend Snippet: Apoptosis, autophagy and inflammation pathway genes evaluated in the present study

Article Snippet: Atg5 , autophagy-related 5 (yeast) , GO:0000045: autophagosome formation , NM_053069 , Mm00504340_m1.

Techniques: Transformation Assay, Binding Assay, Transduction, Full Display Name, Activation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

doi: 10.1038/labinvest.2009.8

Figure Lengend Snippet: Apoptosis, autophagy and inflammation pathway gene expression on cecal ligation and puncture in total liver cells

Article Snippet: Atg5 , autophagy-related 5 (yeast) , GO:0000045: autophagosome formation , NM_053069 , Mm00504340_m1.

Techniques: Gene Expression, Ligation

Journal: Neuron

Article Title: Bassoon Controls Presynaptic Autophagy through Atg5.

doi: 10.1016/j.neuron.2017.01.026

Figure Lengend Snippet: Figure 3. Autophagosomes Accumulate in Periaxonal Soma of DKD Neurons (A) Images of DIV14 mCh-Lamp1/SC (top) and mCh-Lamp1/DKD (bottom) neurons immunostained with Ankyrin-G (arrows) to mark the initial segment of the axon. mCh-Lamp1 puncta are clustered in the periaxonal soma of the DKD neuron. (B) Quantitation of Lamp1 asymmetry in SC and DKD neurons shows an increase in axon-proximal mCh-Lamp1 in DKD neurons. (C) Images of SC and DKD neuronal cell soma (DIV14) labeled with Lysotracker reveals increased axon-proximally located lysosomes in DKD neuron. (D) Quantitation of axon-proximal versus axon-distal Lysotracker puncta in EGFP SC- and EGFP DKD-expressing neurons is shown. (E) Mature autolysosomes, identified as mRFP+/EGFP puncta, were asymmetrically distributed in the soma of tf-LC3 and DKD neurons (DIV14). The addition of 100 nM bafilomycin to DKD neurons (lower panel) increased the number of EGFP+ puncta in DKD neurons. (F) Quantitation of fluorescent puncta from tf-LC3 and DKD neurons treated with DMSO or DMSO and 100 nM bafilomycin A for 3 hr shows a marked increase in mRFP+/EGFP+ puncta after bafilomycin administration. (G) EM micrograph of the cell soma of a DIV14 VAMP2-HRP/DKD neuron containing DAB-labeled vesicular structures as cargo in an autophagic vacuole-like structure. N marks the cell nucleus. DAB-labeled structures were not observed in the soma of any VAMP2-HRP/SC neurons. Scale bars, 10 mm (A, C, and G) and 500 nm (G). Graphs represent mean ± SEM. See Table S1 for means ± SEM, scoring, and n and p values. See also Figure S3 with Movies S1 and S2 and Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human: HeLa, Passage < 30 ATCC CRL-7923 Experimental Models: Organisms/Strains Wildtype rats: Sprague Dawley Charles River, Harlan N/A Wildtype mice: C57Bl6/N Research Institutes for experimental Medicine (FEM) Charité Berlin N/A Bassoon knockout mice Leibniz Institute for Neurobiology Magdeburg: laboratory of Eckart D. Gundelfinger (Davydova et al., 2014) Omnibank ES cell line OST486029 by Lexicon Pharmaceuticals (The Woodlands, TX, USA) Lentivirus Garner and Reimer labs, Stanford University; Virus Core Facility, Charité Berlin N/A Helper-dependent adenovirus (HdAd) Philip Ng, Baylor College of Medicine; (Palmer & Ng 2011) N/A Recombinant DNA FU-SV2-eGFP DKD/SC-W Waites et al., 2013 N/A FU-Vamp2-HRP DKD/SC-W Waites et al., 2013 N/A FU-SV2-eGFP BsnKD-W Waites et al., 2013 N/A FU-eGFP DKD/SC-Wm Waites et al., 2013 N/A FU-mCherry-HA-Ubiquitin K0-W Waites et al., 2013 N/A FU-eGFP-Synapsin1a-W Leal-Ortiz et al., 2008 N/A FU-Synapsin1a Siah1KD-Wm This paper N/A pBsnCC2-eGFP (aa2087-2562, CAA76287.1) Maas et al., 2012 N/A pBsnZF-eGFP (aa1-609, CAA76287.1) Maas et al., 2012 N/A pBsnCD-eGFP(aa1691-3263, CAA76287.1) Maas et al., 2012 N/A pBsnFL-eGFP (aa95-3938, CAA76287.1) Maas et al., 2012 N/A FU-tf-LC3 DKD/SC-Wm This paper ptf-LC3 from Addgene #21073 FU-mRFP-LC3 DKD/SC-Wm This paper mRFP-LC3 from Addgene #21075 FU-mCh-Atg5 DKD/SC-Wm This paper mCh-Atg5 from Addgene #13095 FU-mCh-Lamp1 DKD/SC-Wm This paper pSPAX2 from Addgene #12260 FU-tf-LC3 –Wm This paper ptf-LC3 from Addgene #21073 FU-mRFP-LC3 –Wm This paper mRFP-LC3 from Addgene #21075 FU-mCh-Atg5 –Wm This paper mCh-Atg5 from Addgene #13095 FU-eGFP shAtg5-Wm This paper N/A cDNA rat Lamp1 (NM_012857.2) This paper N/A Lamp1-mCherry This paper pmCherry from Clontech Cat# 632524 FU CFP-SV2 DKD/SC-Wm Noam E. Ziv, Technion, Institute of Technology Haifa Israel N/A FU-CC2v1-eGFP-Synapsin1a-W This paper N/A FU-CC2v1*-eGFP-Synapsin1a-W This paper N/A mRFP-Bassoon* FL Waites et al., 2013 N/A FU-mCherry-Wm This paper N/A pEGFP-N Clontech Cat# 632469 pCMV-VSVg Generous gift of Dr. Theo Palmer; Stanford University N/A Sequence-Based Reagents Atg5 shRNA Obtained from the Stanford Protein and Nucleic Acid facility according to Lee and Gao, 2009 N/A fw 50-gatccccatctgagctatccagacaattcaagag attgtctggatagctcagattttttggaaa-30 Obtained from the Stanford Protein and Nucleic Acid facility according to Lee and Gao, 2009 N/A (Continued on next page) Neuron 93, 897–913.e1–e7, February 22, 2017 e2

Techniques: Quantitation Assay, Labeling, Expressing

Journal: Autophagy

Article Title: The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability.

doi: 10.1080/15548627.2017.1293766

Figure Lengend Snippet: Figure 5. Alterations of autophagosomes and lysosomes as well as changes of autophagy-related proteins after Mn induction in BV2 cells. (A) Representative immunocy- tochemistry of BV2 cells after treatment with Mn for 6 h to determine LC3B protein levels. The LPS and sham groups were used as the positive and negative control groups, respectively. Nuclei were stained with Hoechst. Scale bars: 20 mm. (B) The protein level of ATG5, LC3B, BECN1 and CTSB were analyzed by western blot. The rela- tive band intensities were determined by densitometry. (C) TEM shows the ultrastructure of cells treated with Mn and LPS for 6 h. Arrows indicate autophagosomes, including residual digested material and empty vacuoles; n D 3. Scale bars: 1 mm.

Article Snippet: The membrane was blocked in TRIS-buffered saline (Sigma, T5912) with Tween 20 (Sigma, P9416) containing 5% nonfat dry milk for 2 h and probed with primary antibodies-NLRP3 (Abcam, ab4207), CASP1 (Abcam, ab108362), IL1B (Abcam, ab9722), BECN1 (Cell Signaling Technology, 3738), CTSB (Proteintech, 12216–1-AP), MAP1LC3B/LC3B (Cell Signaling Technology, 2775), ATG5 (Cell Signaling Technology, 12994S), SQSTM1 (Cell Signaling Technology, 8025S), and ACTB (Cell Signaling Technology, 12262) overnight at 4 C and then incubated for 2 h with a horseradish-peroxidase-conjugated antimouse IgG antibody or anti-rabbit IgG diluted 1: 2000 according to the primary antibodies.

Techniques: Negative Control, Staining, Western Blot

Journal: Autophagy

Article Title: The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability.

doi: 10.1080/15548627.2017.1293766

Figure Lengend Snippet: Figure 6. The activation of the NLRP3-CASP1 inflammasome induced by Mn is not restrained by controlling the autophagy elongation stage. BV2 cells were pretreated with Atg5 siRNA for 24 h followed by treatment with 100 mM Mn for 6 h. (A) Representative immunocytochemistry of BV2 cells after treatment with Mn for 6 h to deter- mine LC3 protein levels. Nuclei were stained with Hoechst. Scale bars: 20 mm. (B) The protein level of ATG5, LC3, BECN1 and CTSB were analyzed by western blot. The rel- ative band intensities were determined by densitometry.

Article Snippet: The membrane was blocked in TRIS-buffered saline (Sigma, T5912) with Tween 20 (Sigma, P9416) containing 5% nonfat dry milk for 2 h and probed with primary antibodies-NLRP3 (Abcam, ab4207), CASP1 (Abcam, ab108362), IL1B (Abcam, ab9722), BECN1 (Cell Signaling Technology, 3738), CTSB (Proteintech, 12216–1-AP), MAP1LC3B/LC3B (Cell Signaling Technology, 2775), ATG5 (Cell Signaling Technology, 12994S), SQSTM1 (Cell Signaling Technology, 8025S), and ACTB (Cell Signaling Technology, 12262) overnight at 4 C and then incubated for 2 h with a horseradish-peroxidase-conjugated antimouse IgG antibody or anti-rabbit IgG diluted 1: 2000 according to the primary antibodies.

Techniques: Activation Assay, Immunocytochemistry, Staining, Western Blot

Journal: Autophagy

Article Title: The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability.

doi: 10.1080/15548627.2017.1293766

Figure Lengend Snippet: Figure 7. The release of inflammatory cytokines, such as IL1B induced by Mn is not restrained by controlling the autophagy elongation stage. BV2 cells were pretreated with Atg5 siRNA for 24 h followed by treatment with 100 mM Mn for 6 h. (A) The protein level of mature cleaved IL1B was analyzed by western blot. The relative band intensities were determined by densitometry. (B) The mRNA expression of Il1b was analyzed by qPCR. (C) Levels of IL1B and IL18 in the BV2 cell culture supernatants were measured by ELISA. Statistical analysis was performed with one-way ANOVA. Data are shown as means § SE. P < 0.05 when compared with the untreated group. # P < 0.05 when compared with the Atg5 siRNA group.

Article Snippet: The membrane was blocked in TRIS-buffered saline (Sigma, T5912) with Tween 20 (Sigma, P9416) containing 5% nonfat dry milk for 2 h and probed with primary antibodies-NLRP3 (Abcam, ab4207), CASP1 (Abcam, ab108362), IL1B (Abcam, ab9722), BECN1 (Cell Signaling Technology, 3738), CTSB (Proteintech, 12216–1-AP), MAP1LC3B/LC3B (Cell Signaling Technology, 2775), ATG5 (Cell Signaling Technology, 12994S), SQSTM1 (Cell Signaling Technology, 8025S), and ACTB (Cell Signaling Technology, 12262) overnight at 4 C and then incubated for 2 h with a horseradish-peroxidase-conjugated antimouse IgG antibody or anti-rabbit IgG diluted 1: 2000 according to the primary antibodies.

Techniques: Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability.

doi: 10.1080/15548627.2017.1293766

Figure Lengend Snippet: Figure 8. The activation of the NLRP3-CASP1 inflammasome induced by Mn is not restrained by controlling the autophagy maturation stage. BV2 cells were pretreated with 100 nM BAF for 24 h followed by treatment with 100 mM Mn for 6 h. (A) Representative immunocytochemistry of BV2 cells after treatment with Mn for 6 h to deter- mine LC3B protein levels. Nuclei were stained with Hoechst. Scale bars: 20 mm. (B) The protein level of ATG5, LC3B, BECN1 and CTSB were analyzed by western blot. The relative band intensities were determined by densitometry.

Article Snippet: The membrane was blocked in TRIS-buffered saline (Sigma, T5912) with Tween 20 (Sigma, P9416) containing 5% nonfat dry milk for 2 h and probed with primary antibodies-NLRP3 (Abcam, ab4207), CASP1 (Abcam, ab108362), IL1B (Abcam, ab9722), BECN1 (Cell Signaling Technology, 3738), CTSB (Proteintech, 12216–1-AP), MAP1LC3B/LC3B (Cell Signaling Technology, 2775), ATG5 (Cell Signaling Technology, 12994S), SQSTM1 (Cell Signaling Technology, 8025S), and ACTB (Cell Signaling Technology, 12262) overnight at 4 C and then incubated for 2 h with a horseradish-peroxidase-conjugated antimouse IgG antibody or anti-rabbit IgG diluted 1: 2000 according to the primary antibodies.

Techniques: Activation Assay, Immunocytochemistry, Staining, Western Blot

Journal: Autophagy

Article Title: The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability.

doi: 10.1080/15548627.2017.1293766

Figure Lengend Snippet: Figure 10. Mn enhances NLRP3-CASP1 inflammasome activation and release of inflammatory cytokines, such as IL1B in BV2 cells is blocked by pretreatment with NH4Cl. BV2 cells were treated with 20 mM NH4Cl at the indicated concentrations for 30 min, and subsequently treated with Mn or LPS for 6 h. (A, B) The protein level of ATG5, LC3B, CTSB, cleaved CASP1 and cleaved IL1B were analyzed by western blot. The relative band intensities were determined by densitometry. (C) Levels of IL1B and IL18 in the BV2 cell culture supernatants were measured by ELISA. Statistical analysis was performed with one-way ANOVA. Data are shown as means § SE. P < 0.05 P < 0.01 when compared with the untreated group. #P < 0.05 when compared with the Mn-induced group. & P < 0.05 when compared with the LPS-induced group. @ P < 0.05 when compared with the NH4Cl group.

Article Snippet: The membrane was blocked in TRIS-buffered saline (Sigma, T5912) with Tween 20 (Sigma, P9416) containing 5% nonfat dry milk for 2 h and probed with primary antibodies-NLRP3 (Abcam, ab4207), CASP1 (Abcam, ab108362), IL1B (Abcam, ab9722), BECN1 (Cell Signaling Technology, 3738), CTSB (Proteintech, 12216–1-AP), MAP1LC3B/LC3B (Cell Signaling Technology, 2775), ATG5 (Cell Signaling Technology, 12994S), SQSTM1 (Cell Signaling Technology, 8025S), and ACTB (Cell Signaling Technology, 12262) overnight at 4 C and then incubated for 2 h with a horseradish-peroxidase-conjugated antimouse IgG antibody or anti-rabbit IgG diluted 1: 2000 according to the primary antibodies.

Techniques: Activation Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell & Bioscience

Article Title: Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation

doi: 10.1186/s13578-022-00744-3

Figure Lengend Snippet: Effects of hypoxia and AIF on mitophagy and autophagy in PASMCs. A AIF influenced mitochondrial function by obstructing Tom20 and Lamp2a colocalization, as indicated by the decreased yellow area. Scale bars: 50 μm (n = 4). B PASMCs were exposed to hypoxia for 24 h, and the expression of Pink and Parkin was evaluated by Western blotting (n = 5). C AIF overexpression mitigated the alteration of mitophagy in PASMCs as determined by Mitophagy Keima-Red plasmid transfection. The number of mitochondrial autophagosomes (yellow dots) was calculated (n = 5). Scale bars: 50 μm. D The expression of LC3B-II, P62 and ATG5/7 was evaluated by Western blotting (n = 5). E Autophagic flux and the formation of autophagosomes were detected (n = 6). Scale bars: 50 μm. F Representative electron micrograph of cells after Nor and Hyp treatment. Asterisks represent autophagosomes and arrows represent autolysosomes. Scale bars: 500 nm (n = 3). All data are presented as the means ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; Nor normoxia, Hyp hypoxia, NC negative control

Article Snippet: Antibodies against Cyclin A, Cyclin D, PCNA, UB, P62, ATG5 and ATG7 (Catalog numbers BM1582, BM4272, BM0104, BM4359, BA2849, BA3525 and BA3527) were obtained from Boster (Wuhan, China).

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Standard Deviation, Negative Control

Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 promtoes aggregates deaggregation and clearance via autophagy. (a) Cells were treated with MG132 and immunostained with antibodies to ubiquitin (red) and LC3B-II (green). Arrows indicate ubiquitin-positive aggregates that colocalize with LC3B-positive autophagosomes. Scale bars: 10 μm. (b) Confocal images of TRIM44[OE-CON] and TRIM44[OE] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA (10 mM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (c) Confocal images of TRIM44[KD-CON] and TRIM44[KD] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM). Arrows indicate cells with remaining aggregates. Scale bars: 10 µm. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (d, e) Confocal images of WT or ATG5 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (d). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (e). (f, g) Confocal images of WT or BECN1 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells were treated with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (f). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of BECN1 and TRIM44 were assayed by western blot (g). (h, i) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. The status of aggregates after MG132 treatment was quantified in the histogram. Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (h). (j) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) together with MG132 (5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm.

Article Snippet: Anti-TRIM44 polyclonal antibody (Proteintech Group, 11,511-1-AP); anti-Ub antibody (Biolegend, 646,301); anti-mCherry antibody (ThermoFisher, PA5-34,974); anti-VIM antibody (ThermoFisher, MA5-11,883); anti-NFE2L2/NRF2 antibody (ThermoFisher, PA5-27,882); anti-20S proteasome antibody (MilliporeSigma, ST1049); anti-TUBG/γ-Tubulin antibody (MilliporeSigma, T5326); anti-ATG5 antibody (Novus Biologicals, NBP2-24,389); Anti-GFP antibody (Santa Cruz Biotechnology, sc-9996); anti-ACTB/β-Actin antibody (Santa Cruz Biotechnology, sc-47,778), anti-HA antibody (Santa Cruz Biotechnology, sc-805); anti-BECN1 antibody (Cell Signaling Technology, 4122); anti-HDAC6 antibody (Cell Signaling Technology, 7558); anti-LC3B antibody (Cell Signaling Technology, 3868); anti-SQSTM1/p62 antibody (Cell Signaling Technology, 88,588); anti-cleaved PARP antibody (Cell Signaling Technology, 5625); Alexa Fluor 594 donkey anti rabbit IgG(H + L) (Invitrogen, A21207); Alexa Fluor 546 goat anti mouse IgG(H + L) (Invitrogen, A11003).

Techniques: Ubiquitin Proteomics, Transfection, Staining, Western Blot