Journal: Autophagy

Article Title: Zika virus NS2A protein induces the degradation of KPNA2 (karyopherin subunit alpha 2) via chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1823122

Figure Lengend Snippet: List of primers used in this study

Article Snippet: The primary mouse monoclonal antibodies against KPNA1 (Santa Cruz Biotechnology, sc-101,292), KPNA2 (Santa Cruz Biotechnology, sc-55,537), hemagglutinin (HA) tag (ThermoFisher Scientific, 26,183), GFP (Biolegend, 75,818–584), HSPA8/HSC70 (Santa Cruz Biotechnology, sc-7298), ubiquitin (Santa Cruz Biotechnology, sc-8017), GAPDH (Santa Cruz Biotechnology, sc-365,062), and TUBB1/β-tubulin (Sigma, T7816), and rabbit polyclonal antibodies against ZIKV NS4B (GeneTex, GTX133311), ZIKV E (GeneTex, GTX133314), NS2B (GeneTex, GTX133318), NS5 (GeneTex, GTX133329) and LAMP2A (Boster, M01573) were used in this study.

Techniques:

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet: Phenylpyruvate upregulated NLRP3 palmitoylation by binding to the PPT1 protein (A) Schematic view of NLRP3 (full length) and the location of the S-palmitoylation sites. (B) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs treated with or without palmitic acid (n = 3). (C) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP at the indicated concentrations (0, 50, 100, and 150 μM) for 24 h (n = 3). (D) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP (100 μM) for 0, 8, 16, and 24 h (n = 3). (E) Immunostaining of the location of LAMP2 (indicating lysosomes), PPT1, and NLRP3 in macrophages. The nuclei were stained with Hoechst dye. Scale bar, 10 μm. (F and G) Immunoprecipitation (IP) and immunoblot analysis were used to detect the endogenous NLRP3 and PPT1 association in macrophages (n = 3). (H) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs with Ppt1 knockdown (n = 3). (I) Mouse embryo fibroblasts (MEFs) were transfected with WT NLRP3 or the indicated NLRP3 mutants for 24 h with or without knockdown of Ppt1. ABE assay and immunoblot analysis showing palmitoylation levels of the indicated NLRP3 mutants (n = 3). (J) ABE assay and immunoblot analysis were used to determine NLRP3 palmitoylation levels in BMDMs treated with increasing phenylpyruvate concentrations (n = 3). (K) Determination of NLRP3 palmitoylation levels in BMDMs treated with 400 μM phenylpyruvate and then transfected with the Ppt1 WT plasmid or the MUT plasmid for 24 h (n = 3). (L) BMDMs were treated with 400 μM phenylpyruvate with or without 100 μM 2BP and then transfected with or without the Ppt1 MUT plasmid. ABE assay and immunoblot analysis determining NLRP3 palmitoylation levels in the indicated cells (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Binding Assay, Western Blot, Expressing, Immunostaining, Staining, Immunoprecipitation, Knockdown, Transfection, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet:

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Recombinant, Transfection, Lysis, cDNA Synthesis, Real-time Polymerase Chain Reaction, BIA-KA, Enzyme-linked Immunosorbent Assay, Software

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate suppressed osteoclast differentiation through autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast markers TRAF6, c-Fos, MMP-9, MMP-14, and CTSK ( d ) and the autophagic proteins Beclin1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts received different treatments for 5 days. (n = 3, mean ± SD, ** p < 0.01, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate inhibited osteoclast differentiation through NF-κB-pathway-dependent autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast marker CTSK, the proteins central to the NF-κB pathway (p-IKKα/β, IκBα, and p65) ( d ), and the autophagic proteins Beclin-1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts were treated with Bay 11-7082 for 5 days. ( f ) Western blot assay and relative protein expression of the proteins central to the NF-κB pathway after pre-osteoclasts were treated with SR for 5 days. (n = 3, mean ± SD, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing, Marker

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate regulated the expression of autophagic proteins in rats. ( a ) Immunohistochemical staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. ( b ) The mean optical density (MOD) of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on days 3, 7, and 14 in different groups. (n = 5, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001). ( c ) Immunofluorescence staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. The white dashed lines indicate the margin of the tooth root.

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

doi: 10.1371/journal.pone.0031232

Figure Lengend Snippet: ( A ) Representative fluorescent images of GAPDH-HT 21 h after labeling with TMR-HT ligand in HeLa cells transfected with nontargeting-siRNA (left) or LAMP2A-siRNA (right). Bar = 20 µm. ( B,C ) Quantitative analyses of GAPDH-HT lysosomal translocation in cells transfected with nontargeting (Non)- and LAMP2A-siRNA. ( B ) Cells having more than 5 dots of GAPDH-HT were classified as GAPDH-HT dot-positive cells. We counted the number of GAPDH-HT dot-positive cells in 50–70 GAPDH-HT-expressing cells. ( C ) We assessed the number of GAPDH-HT dots per cell. The percentage of GAPDH-HT dot-positive cells and the number of GADPH-HT dots per cell were significantly decreased by siRNA-mediated LAMP2A-knockdown. ** p<0.001 vs cells treated with nontargeting-siRNA (unpaired t -test, n = 16 in B , n = 57 for nontargeting-siRNA and n = 74 for LAMP2A-siRNA in C ). ( D ) Representative fluorescent images of GAPDH-HT taken 21 h after labeling with the TMR-HT ligand in HeLa cells treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), serum free medium (0.1% DMSO, 0.1% methanol, center upper), H 2 O 2 (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower), SB202190 (20 µM, left center) or cycloheximide (CHX; 20 µg/ml, right lower). Bar = 20 µm. ( E,F ) Quantitative analyses of GAPDH-HT lysosomal translocation in cells treated with CMA activators or inhibitors the percentages of GAPDH-HT dot-positive cells ( E ) and the number of GAPDH-HT dots per cell ( F ). Percentages of GAPDH-HT dot-positive cells and the numbers of GAPDH-HT dots per cell were significantly increased by CMA activators (serum free medium, H 2 O 2 and MPA), while they were significantly decreased by CMA inhibitors (SB202190 and CHX). * p<0.01, ** p<0.001 vs cells treated with vehicle (unpaired t -test, n = 12 for cells treated with vehicle, n = 8 for cells treated with CMA activators and inhibitors in E , n = 96 for vehicle, n = 61 for serum free, n = 45 for H 2 O 2 , n = 58 for MPA, n = 52 for SM202190 and n = 41 for CHX in F ).

Article Snippet: Nontargeting-siRNA was used as a negative control of siRNA transfection. siRNAs against human LAMP2A (sense: 5′-GGCAGGAGUACUUAUUCUAGU-3′ , antisense: 5′-UAGAAUAAGUACUCCUGCCAA-3′ ) and human Atg5 (sense: 5′-CACUUUCAGAAGGUUAUGAGA-3′ , antisense: 5′-UCAUAACCUUCUGAAAGUGCU-3′ ) were constructed by Hayashi-kasei (Osaka, Japan). siRNA (50 pmol) was transfected to HeLa cells 1 day after cell spread using Lipofectamine RNAiMAX.

Techniques: Labeling, Transfection, Translocation Assay, Expressing, Knockdown

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MANF Inhibits α -Synuclein Accumulation through Activation of Autophagic Pathways

doi: 10.1155/2022/7925686

Figure Lengend Snippet: MANF inhibited the accumulation of SNCA WT in PD cellular model by CMA activation. (a)–(d) Effects of MANF on the levels of SNCA WT , Lamp-2A, and Hsc70. SNCA WT SH-SY5Y cells were treated with MANF (250, 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively; then, the protein levels were detected by western blot analysis. (e, f) Effects of MANF on the interaction between SNCA WT and Hsc70. SNCA WT SH-SY5Y cells were treated with MANF (500 ng/ml) and Dox (600 ng/ml) for 24 h. Cell lysates were immunoprecipitated with anti-SNCA, and the precipitated proteins were analyzed by immunoblotting with anti-Hsc70. (g, h) SNCA WT SH-SY5Y cells were treated with Lamp-2A RNAi 1-3 for 48 h. The level of LAMP-2A was detected by western blot analysis. (i, j) Lamp-2A knocked down partly abolished MANF-induced SNCA WT clearance. SNCA WT SH-SY5Y cells were treated with Lamp-2A RNAi 2 for 24 h, followed by the incubation of MANF (500 ng/ml) and Dox (600 ng/ml) for another 24 h. The levels of LAMP-2A and SNCA WT were detected by western blot analysis. Data were expressed as mean ± SD from three independent experiments. ## P < 0.01 vs. control group; $ P < 0.05 vs. NC group; ∗ P < 0.05 and ∗∗ P < 0.01 vs. Dox-treated group; & P < 0.05 vs. combined treatment with negative control Lamp-2A RNAi plasmid, MANF, and Dox group.

Article Snippet: The human SNCA WT SH-SY5Ycells were plated into 12-well plates at the density of 2 × 10 5 cells/well and then transfected with LAMP-2A ShRNA or Lenti-mCherry-eGFP-LC3B plasmid (both from Genechem, Shanghai, China) using transfection reagent lipofectamine 3000 (Invitrogen, Shanghai, China), according to the manufacturer's protocol.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Incubation, Negative Control, Plasmid Preparation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MANF Inhibits α -Synuclein Accumulation through Activation of Autophagic Pathways

doi: 10.1155/2022/7925686

Figure Lengend Snippet: Effects of Nrf2 on the early activation of macroautophagy by MANF. (a, b) SNCA WT SH-SY5Y cells were treated with Lamp-2A-RNAi 2 plasmid for 24 h, followed by incubation with MANF (500 ng/ml) and Dox (600 ng/ml) for another 24 h. The levels of LC3, Beclin-1, and P62 were detected by western blot analysis. (c, d) SNCA WT SH-SY5Y cells were treated with MANF (500 ng/ml) and Dox (600 ng/ml) for another 24 h or 48 h, respectively; cell lysates were immunoblotted by anti-Nrf2 antibody. (e, f) SNCA WT SH-SY5Y cells were treated with Lamp-2A-RNAi 2 plasmid for 24 h, followed by incubation with MANF (500 ng/ml), ML385 (5 μ M), and Dox (600 ng/ml) for another 24 h. The levels of LAMP-2A, Nrf2, LC3, Beclin-1, and P62 were detected by western blot analysis. Data were expressed as mean ± SD from three independent experiments. # P < 0.05 vs. control group; ∗ P < 0.05 vs. Dox-treated group; & P < 0.05 vs. combined treatment with MANF and Dox group.

Article Snippet: The human SNCA WT SH-SY5Ycells were plated into 12-well plates at the density of 2 × 10 5 cells/well and then transfected with LAMP-2A ShRNA or Lenti-mCherry-eGFP-LC3B plasmid (both from Genechem, Shanghai, China) using transfection reagent lipofectamine 3000 (Invitrogen, Shanghai, China), according to the manufacturer's protocol.

Techniques: Activation Assay, Plasmid Preparation, Incubation, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MANF Inhibits α -Synuclein Accumulation through Activation of Autophagic Pathways

doi: 10.1155/2022/7925686

Figure Lengend Snippet: MANF inhibited the accumulation of SNCA A53T in PD cellular model. (a, b) SNCA A53T SH-SY5Y cells were treated with MANF (0, 250, 500, 1000, or 2000 ng/ml) and Dox (600 ng/ml) for 24 h, and cell lysates were immunoblotted by anti-SNCA antibody. (c, d) SNCA A53T SH-SY5Y cells were treated with MANF (500 ng/ml) and Dox (600 ng/ml) for 24 h, and the level of SNCA was detected by immunofluorescence staining. Scale bar is 50 μ m. (e)–(h) Effects of MANF on the levels of SNCA, Lamp-2A, and Hsc70. SNCA A53T SH-SY5Y cells were treated with MANF (250 and 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively; then, the protein levels were detected by western blot analysis. Data were expressed as mean ± SD from three independent experiments. ## P < 0.01 vs. control group; ∗ P < 0.05, ∗∗ P < 0.01 vs. Dox-treated group.

Article Snippet: The human SNCA WT SH-SY5Ycells were plated into 12-well plates at the density of 2 × 10 5 cells/well and then transfected with LAMP-2A ShRNA or Lenti-mCherry-eGFP-LC3B plasmid (both from Genechem, Shanghai, China) using transfection reagent lipofectamine 3000 (Invitrogen, Shanghai, China), according to the manufacturer's protocol.

Techniques: Immunofluorescence, Staining, Western Blot