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: The Journal of Cell Biology

Article Title: Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy

doi: 10.1083/jcb.201605065

Figure Lengend Snippet: The role of autophagy in morphine-mediated synaptic alterations. Representative Western blot (A) and quantification of autophagy mediators (Beclin1, ATG5, p62, and LC3-II; B) in the hippocampal lysates from mouse injected with saline or morphine ( n = 3 to 4/group). *, P < 0.05; **, P < 0.01 versus saline group using Student’s t test. Representative time course Western blot (C) and quantification of LC3-II (D) in primary neurons exposed to morphine. Representative Western blot (E) and quantification of Beclin1, ATG5, p62, and LC3-II (F) in the cell lysates from neurons pretreated with naltrexone for 1 h followed by 24-h morphine treatment. Representative confocal images (G) and quantification of GFP-LC3 puncta (H) of primary hippocampal neurons expressing GFP-LC3 exposed to morphine for 24 h. Bar, 10 µm. **, P < 0.01 versus saline group using Student's t test. Representative Western blot (I) and quantification of LC3-II (J) in the cell lysates of neurons treated with bafilomycin for 4 h after 24-h exposure to morphine. Representative confocal images of GFP-expressing primary rat hippocampal neurons treated with wortmannin (500 µM) for 1 h followed by morphine exposure for 24 h and stained with vGlut1 and GAD65 (K) and quantifications of spine density and excitatory and inhibitory synapses (L). Bar, 5 µm. Representative confocal images of neurons expressing PGK-GFP-scramble or PGK-GFP-shATG7 vector in the presence or absence of morphine for 24 h and stained with vGlut1 and GAD65 (M) and quantifications of spine density and excitatory and inhibitory synapses (N). Bar, 5 µm. Quantification of Western blot results were all normalized to β-actin. Each set of in vitro results was quantified upon four independent experiments. All data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 versus control group; # , P < 0.05; ## , P < 0.01 versus morphine group using one-way ANOVA with post hoc test (except B and H).

Article Snippet: The Western blots were then probed with respective antibodies recognizing the vGlut1 (1:5,000; catalog no. AB5905, lot no. RRID:AB_2301751; EMD Millipore), PSD95 (1:1,000; catalog no. ab18258; lot no. RRID:AB_444362; Abcam), GAD65 (1:1,000; catalog no. NBP1-33284, lot no. RRID:AB_10004060; Novus Biologicals), gephyrin (1:250; catalog no. 610584, lot no. RRID:AB_397929; BD), BIP (1:1,000; catalog no. 610978, lot no. RRID:AB_398291; BD), pPERK (1:250; catalog no. sc-32577, lot no. RRID:AB_2293243; Santa Cruz Biotechnology, Inc.), PERK (1:250; catalog no. sc-13073, lot no. RRID:AB_2230863; Santa Cruz Biotechnology, Inc.), IRE1α (1:500; catalog no. sc-20790, lot no. RRID:AB_2098712; Santa Cruz Biotechnology, Inc.), ATF6 (1:1,000; catalog no. ab37149, lot no. RRID:AB_725571; Abcam), Beclin1 (1:1,000; catalog no. sc-11427, lot no. RRID:AB_2064465; Santa Cruz Biotechnology, Inc.), ATG5 (1:1,000; catalog no. NB110-53818, lot no. RRID:AB_828587, Novus Biologicals), p62 (1:1,000; catalog no. 50–507-55, lot no. RRID: AB_2571590; Thermo Fisher Scientific), LC3 (1:2,000; catalog no. NB100-2220, lot no. RRID:AB_10003146; Novus Biologicals), and β-actin (1:5,000; catalog no. A1978, lot no. RRID:AB_476692; Sigma-Aldrich).

Techniques: Western Blot, Injection, Saline, Expressing, Staining, Plasmid Preparation, In Vitro, Control

Journal: The Journal of Cell Biology

Article Title: Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy

doi: 10.1083/jcb.201605065

Figure Lengend Snippet: Morphine-mediated autophagy in hippocampal neurons: Role of oxidative and ER stress. Representative Western blot (A) and quantification of autophagy mediators (Beclin1, ATG5, p62, and LC3-II; B) in the cell lysates of neurons pretreated with PBN or apocynin for 1 h followed by 24-h morphine treatment. (C) H 2 DCFDA assay of neurons pretreated with 3-MA (50 µM) for 30 min followed by morphine exposure. Representative Western blot (D) and quantification of Beclin1, ATG5, p62, and LC3-II (E) in the cell lysates of neurons pretreated with 4-PBA or salubrinal (100 µM) for 1 h followed by 24-h morphine exposure. Representative Western blot (F) and quantification of BIP, pPERK, IRE1α, and ATF6 (G) in the cell lysates of neurons pretreated with wortmannin or 3-MA for 1 h followed by 24-h morphine exposure. Quantification of Western blot results were all normalized to β-actin, except that pPERK was normalized to PERK. Each set of our results was quantified upon four independent experiments. All data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 versus control/saline group; # , P < 0.05; ## , P < 0.01 versus morphine group using one-way ANOVA with post hoc test.

Article Snippet: The Western blots were then probed with respective antibodies recognizing the vGlut1 (1:5,000; catalog no. AB5905, lot no. RRID:AB_2301751; EMD Millipore), PSD95 (1:1,000; catalog no. ab18258; lot no. RRID:AB_444362; Abcam), GAD65 (1:1,000; catalog no. NBP1-33284, lot no. RRID:AB_10004060; Novus Biologicals), gephyrin (1:250; catalog no. 610584, lot no. RRID:AB_397929; BD), BIP (1:1,000; catalog no. 610978, lot no. RRID:AB_398291; BD), pPERK (1:250; catalog no. sc-32577, lot no. RRID:AB_2293243; Santa Cruz Biotechnology, Inc.), PERK (1:250; catalog no. sc-13073, lot no. RRID:AB_2230863; Santa Cruz Biotechnology, Inc.), IRE1α (1:500; catalog no. sc-20790, lot no. RRID:AB_2098712; Santa Cruz Biotechnology, Inc.), ATF6 (1:1,000; catalog no. ab37149, lot no. RRID:AB_725571; Abcam), Beclin1 (1:1,000; catalog no. sc-11427, lot no. RRID:AB_2064465; Santa Cruz Biotechnology, Inc.), ATG5 (1:1,000; catalog no. NB110-53818, lot no. RRID:AB_828587, Novus Biologicals), p62 (1:1,000; catalog no. 50–507-55, lot no. RRID: AB_2571590; Thermo Fisher Scientific), LC3 (1:2,000; catalog no. NB100-2220, lot no. RRID:AB_10003146; Novus Biologicals), and β-actin (1:5,000; catalog no. A1978, lot no. RRID:AB_476692; Sigma-Aldrich).

Techniques: Western Blot, Control, Saline

Journal: The Journal of Cell Biology

Article Title: Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy

doi: 10.1083/jcb.201605065

Figure Lengend Snippet: Protective role of PDGF-BB in morphine-mediated synaptic alterations. Representative confocal images of GFP-expressing primary rat hippocampal neurons exposed to morphine followed by treatment with PDGF-BB (20 ng/ml) and stained with vGlut1 and GAD65 (A) and quantifications of spine density and excitatory and inhibitory synapses (B). Bar, 5 µm. Representative traces of whole-cell voltage-clamp recording showing mEPSC (C), mIPSC (E), mean mEPSC frequencies (D), and mean frequencies of mIPSCs (Hz; F) in primary rat neurons (DIV 19–21) treated with combinations of saline, morphine, and PDGF-BB as indicated. (G) ROS generation in neurons exposed to morphine and treated with PDGF-BB. Representative Western blot (H) and quantification of BIP, pPERK, IRE1α, and ATF6 (I) in the cell lysates of neurons exposed to morphine followed by treatment with PDGF-BB (20 ng/ml) for an additional 24 h. Representative Western blot (J) and quantification of Beclin1, ATG5, p62, and LC3-II (K) in the cell lysates of neurons exposed to morphine for 24 h followed by PDGF-BB (20 ng/ml) for additional 24 h. Quantification of Western blot results were all normalized to β-actin except that pPERK was normalized to PERK. Each set of our results was quantified upon four independent experiments. All data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 versus control/saline group; # , P < 0.05; ## , P < 0.01 versus morphine group using one-way ANOVA with post hoc test.

Article Snippet: The Western blots were then probed with respective antibodies recognizing the vGlut1 (1:5,000; catalog no. AB5905, lot no. RRID:AB_2301751; EMD Millipore), PSD95 (1:1,000; catalog no. ab18258; lot no. RRID:AB_444362; Abcam), GAD65 (1:1,000; catalog no. NBP1-33284, lot no. RRID:AB_10004060; Novus Biologicals), gephyrin (1:250; catalog no. 610584, lot no. RRID:AB_397929; BD), BIP (1:1,000; catalog no. 610978, lot no. RRID:AB_398291; BD), pPERK (1:250; catalog no. sc-32577, lot no. RRID:AB_2293243; Santa Cruz Biotechnology, Inc.), PERK (1:250; catalog no. sc-13073, lot no. RRID:AB_2230863; Santa Cruz Biotechnology, Inc.), IRE1α (1:500; catalog no. sc-20790, lot no. RRID:AB_2098712; Santa Cruz Biotechnology, Inc.), ATF6 (1:1,000; catalog no. ab37149, lot no. RRID:AB_725571; Abcam), Beclin1 (1:1,000; catalog no. sc-11427, lot no. RRID:AB_2064465; Santa Cruz Biotechnology, Inc.), ATG5 (1:1,000; catalog no. NB110-53818, lot no. RRID:AB_828587, Novus Biologicals), p62 (1:1,000; catalog no. 50–507-55, lot no. RRID: AB_2571590; Thermo Fisher Scientific), LC3 (1:2,000; catalog no. NB100-2220, lot no. RRID:AB_10003146; Novus Biologicals), and β-actin (1:5,000; catalog no. A1978, lot no. RRID:AB_476692; Sigma-Aldrich).

Techniques: Expressing, Staining, Saline, Western Blot, 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

Journal: Cells

Article Title: Studying Autophagy in Zebrafish

doi: 10.3390/cells6030021

Figure Lengend Snippet: List of antibodies ever used to detect autophagy-related proteins in zebrafish. (Catalogue numbers listed in italics have been used for immunostaining too).

Article Snippet: Atg5 , Novus biologicals , NB110-53818 , [ , ] .

Techniques: Immunostaining

Journal: Veterinary research

Article Title: Incomplete autophagy promotes the proliferation of Mycoplasma hyopneumoniae through the JNK and Akt pathways in porcine alveolar macrophages.

doi: 10.1186/s13567-022-01074-5

Figure Lengend Snippet: Figure 1 M. hyopneumoniae infection increases the formation of autophagosome-like vesicles and the expression of autophagy-related proteins in porcine alveolar macrophages. A Autophagosome-like vesicles (arrows) were observed in M. hyopneumoniae-negative (a) and M. hyopneumoniae-positive (b) PAMs by TEM. Ten M. hyopneumoniae-negative and 10 M. hyopneumoniae-positive PAMs were assessed. B Autophagosome-like vesicles (arrows) were observed in mock-infected (a) or AH-infected (b) 3D4/21 cells by TEM. Ten uninfected and 10 AH-infected 3D4/21 cells were assessed. C Results of Western blotting showing the expression of autophagy-related proteins (LC3-II, Atg5, Atg12-Atg5, and Beclin 1) and Mhp366 in 30 M. hyopneumoniae-positive and 10 M. hyopneumoniae-negative PAMs collected from pigs. D Results of Western blotting showing the expression of autophagy-related proteins (LC3-II, Atg5, Atg12-Atg5, and Beclin 1) and P97 protein in 3D4/21 cells after mock infection or AH infection. Mean values from 3 independent experiments are presented. Between-group differences were assessed using a t test; *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: Polyclonal antibodies against ATG5 (NBP2-24389), ATG5 (NB11053818), Beclin 1 (NB110-87318), and p62 (NBP1-48320) were purchased from Novus Biologicals (Shanghai, China).

Techniques: Infection, Expressing, Western Blot