Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural comparison of Cas12k-transposon recruitment and Cas12k-TnsC non-productive complexes, related to Figures 1 , , and (A) Cryo-EM density maps of the Cas12k-transposon recruitment complex (top) and the Cas12k-TnsC non-productive complex (bottom). Side views and structural superpositions are shown. Proteins are shown in surface representation. The DNA in the transposon recruitment complex is bent by ∼56° relative to the non-productive complex. (B) Atomic models, shown in surface representation, of the Cas12k-transposon recruitment complex (top) and the Cas12k-TnsC non-productive complex (bottom).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: R-loop completion upon complex assembly (A) Detailed views of the R-loop structure in the Cas12k-transposon recruitment complex, comprised of the crRNA portion of the single guide RNA (red cartoon backbone), the TS (blue cartoon backbone), and the NTS (dark gray cartoon backbone). Only the REC lobe, the RuvC domain, and the bridging helix (BH) of Cas12k are shown for clarity. (B) Detailed views of the R-loop structure in the Cas12k-sgRNA-target-DNA complex (PDB: 7PLA ). (C) Zoomed-in view of the PAM-distal end of the R-loop in the Cas12k-transposon recruitment complex. Density corresponding to nucleic acids is shown (contour level of 7.7 σ). TniQ is depicted as surface representation. See also Figure S4 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural rearrangements in Cas12k and guide RNA upon R-loop completion, related to Figure 2 (A) Structural models of the Cas12k-sgRNA-target DNA complex (PDB: 7PLA , top) and the Cas12k-transposon recruitment complex bottom), shown in the same orientation. Domain architecture of Cas12k is shown below each model. REC, recognition lobe. WED, wedge domain. PI, PAM interacting domain. BH, bridging helix. TS, target DNA strand; NTS, non-target DNA strand. (B) Structural superpositions of the RuvC and BH domains in the Cas12k-sgRNA-target DNA and Cas12k-transposon recruitment complexes. (C) Structural superposition of the tracrRNA part of the sgRNA in the Cas12k-sgRNA-target DNA (gray) and the Cas12k-transposon recruitment complex (orange).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: TniQ recognizes tracrRNA and completed R-loop (A) Overview of TniQ in the Cas12k-transposon recruitment complex, depicting interfaces with the tracrRNA (orange) and the RNA:DNA heteroduplex formed by the crRNA (red) and the TS (blue). NTS is colored in dark gray. The N and C termini of TniQ are indicated. (B) Close-up view of key tracrRNA-interacting residues of TniQ. (C) Site-specific transposition activity in E. coli of ShCAST systems containing structure-based mutations in the tracrRNA or the tracrRNA-interacting interface in TniQ, as determined by droplet digital PCR (ddPCR) analysis. Data are presented as mean ± SD (n = 3 biologically independent replicates). (D) Detailed view of R-loop recognition by TniQ. (E) Site-specific transposition activity in E. coli of ShCAST systems containing structure-based mutations in the R-loop recognition interface of TniQ. Data are presented as mean ± SD (n = 3 biologically independent replicates).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Activity Assay, Digital PCR

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Cryo-EM analysis of TniQ-capped TnsC filament, related to Figures 5 and and STAR Methods (A) Cryo-EM image processing workflow for the TniQ-capped TnsC filament complex. (B) Fourier Shell Correlation (FSC) of TnsC-DNA-TniQ reconstruction from two independently refined half-maps. The gold-standard cut-off (FSC = 0.143) is marked with a blue line. (C) Final electron density map colored according to the local resolution. (D) Fourier Shell Correlation (FSC) of the reconstruction from two independently refined half-maps.

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural comparisons of TniQ-capped TnsC filament and Cas12k-transposon recruitment complex, related to Figures 5 and and Table S1 (A) Side and top views of the TniQ-capped TnsC filament. Proteins are shown in surface representation. (B) Side and top views of the Cas12k-transposon recruitment complex, with TniQ shown in the same orientation as TniQ1 in (A). In the top view, Cas12k, S15 and tracrRNA are omitted to visualize the contacts between TniQ and TnsC. (C) Structural overlay of three consecutive TnsC protomers (TnsC1-TnsC3) in the Cas12k-transposon recruitment complex (colored protomers, white DNA) and in the TniQ-capped TnsC filament (gray protomers and DNA) and the associated DNA (shown in stick representation).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: TnsC assembly on PAM-distal end of R-loop DNA (A) Overview of guide-target R-loop structure within the Cas12k-transposon recruitment complex. TS (blue) and NTS (dark gray) are shown in cartoon format. Only the crRNA portion of the single-guide RNA (red) is shown. Proteins are shown in surface representation. Residues 132–254 of Cas12k and the TnsC2 and TnsC3 protomers are omitted from view for clarity. (B) Zoomed-in view of target DNA-binding residues of TniQ. (C) Zoomed-in view of the DNA-binding residues in the TnsC1 protomer. (D) Comparison of DNA binding modes of consecutive TnsC protomers (TnsC1–TnsC3) in the Cas12k-transposon recruitment complex (left) and in the TniQ-capped TnsC filament (right). See also Figure S5 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Binding Assay

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: S15 promotes Cas12k-transposon recruitment complex assembly and transposition activity (A) Zoomed-in view of S15 binding in the Cas12k-recruitment complex. (B) Co-precipitation of TnsC and TniQ in presence or absence of S. hofmanni S15 (ShS15) or E. coli S15 (EcS15) by immobilized Cas12k-sgRNA-target DNA complex. (C) In vitro transposition activity of purified ShCAST components in the absence or presence of EcS15 (wild-type or mutant), ShS15, and Homo sapiens RPS13 (HsS13) proteins, as determined by ddPCR analysis. Data are presented as mean ± SD (n = 4 independent replicates). Statistical analysis was conducted using unpaired two-tailed t-tests. P-values: ∗ p < 0.05; ∗∗ p < 0.01; ns, not significant. See also Figure S7 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Activity Assay, Binding Assay, In Vitro, Purification, Mutagenesis, Two Tailed Test

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Interactions and conservation of the ribosomal protein S15, related to Figure 6 (A) Zoomed-in view of E. coli S15 interactions with the tracrRNA and crRNA:TS-DNA duplex in the Cas12k-transposon recruitment complex. (B) Zoomed-in view of S15 contacts with the Cas12k REC2 domain. (C) Co-precipitation of E. coli S15 (EcS15) wild-type and mutant, S. hofmanni S15 (ShS15) and Homo sapiens RPS13 (HsS13) proteins by immobilized Cas12k-sgRNA complex. (D) Sequence alignment of the ribosomal proteins EcS15, ShS15 and HsS13. (E) Zoomed-in views of EcS15 interactions with tracrRNA in the Cas12k-transposon recruitment complex (left), 16S rRNA in the E. coli ribosome (middle; PDB: 6Q97 ), and a superposition of both focused on S15 (right). (F) Structural models of the Cas12k-transposon recruitment complex (left), Cas12e-sgRNA-target DNA complex (middle; PDB: 6NY2 ), and their superposition focused on S15 (right).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Mutagenesis, Sequencing

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Mechanism of RNA-guided assembly in type V CASTs Mechanistic model for the recruitment of the transposition machinery by the RNA-guided Cas12k complex in type V-K CASTs. Cas12k in association with a crRNA-tracrRNA dual guide RNA initially binds target DNA to form a partial R-loop structure. Full R-loop formation occurs upon recruitment of S15, TniQ, and TnsC. TniQ recognizes specific regions of the tracrRNA and primes polymerization of a TnsC filament by bridging the first two TnsC protomers. The ribosomal protein S15 facilitates productive complex assembly by interacting with tracrRNA and Cas12k. The resulting TnsC filament provides a recruitment platform for TnsB, which triggers TnsC depolymerization to expose the insertion site and catalyzes transposon DNA insertion.

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet:

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Recombinant, Mass Spectrometry, Electron Microscopy, Plasmid Preparation, Clone Assay, Software

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: ELISA platform for assessment of uEVs (A) Schematic illustration of sandwich ELISA using Tim4. (B) Expression of MGAM, MUC1, and CD9 in nephron segments. The expression pattern was visualized by kidney cell explorer and by fluorescent immunohistochemistry using human kidney tissue. The numbers above the figure are the same as in Figure 1 G. Scale bars, 100 μm. See also Figures S4 and .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Expressing, Immunohistochemistry

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Application of expression signature of uEVs for diagnosis of CKD (A) Comparison of MUC1 and MGAM levels (absorbance at 450 nm) measured by customized ELISA in samples from healthy controls and patients with CKD. G1 indicates CKD patients with eGFR ≥90 and G2-5 with <90 mL/min/1.73m 2 . (B and C) ROC curve for distinguishing patients with decreased renal function (eGFR <60 (B), eGFR <90 (C)) from healthy controls by logistic regression. (D) ROC curve for distinguishing patients with CKD having normal eGFR (≥90) from healthy controls by logistic regression. (E) Box and beeswarm plot of the assay value of MGAM/MUC1 (the expression of MGAM divided by that of MUC1) in patients with CKD having each renal function and healthy controls in the discovery and validation cohorts. (F) ROC curve for distinguishing patients with decreased renal function (eGFR <90) from healthy controls by logistic regression using combinations of MGAM/MUC1, urine creatinine (uCr), urine albumin (uAlb), and L-FABP. (G) The change in eGFR in follow-up period in two groups (MGAM/MUC1 below 0.35 and the others). Boxes in boxplots indicate the 25th and 75th percentiles, and the horizontal lines inside the boxes indicate the median. Bars indicate the 10th and 90th percentiles. The data were compared using the two-tailed Mann–Whitney U test. See also Figures S6–S8 .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Expressing, Biomarker Discovery, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

doi: 10.1038/cddis.2010.36

Figure Lengend Snippet: Cofilin translocates to the mitochondria upon oxidative stress. ( a ) Primary human T cells were incubated with (lower panel) or without (upper panel) 50 μ M H 2 O 2 . Thereafter, cells were stained for cofilin (red) or mitochondria (MitoTracker, green) and analyzed via confocal laser scan microscopy. Merge displays the digital overlay of red and green fluorescence. The figure is representative of three independent experiments. ( b ) For cryo-immunogold electron microscopy, primary human T cells were either left untreated (i and iii) or treated with H 2 O 2 (ii and iv) and subsequently fixed with 2% PFA for 10 min. Cells were stained with cofilin antiserum combined with protein A labeled with 15 nm gold particles. Shown are two example pictures taken from two independent experiments (M, mitochondria; N, nucleus). ( c , d ) The colocalization of cofilin and mitochondria was evaluated by the calculation of a similarity score of the two probes from untreated (gray histogram) and H 2 O 2 -treated (black lined histogram) PBT using MIFC. The histogram shows the distribution of the similarity within the whole-cell population as in conventional flow cytometry (up to 10 000 cells). A score of 1 indicates that the two probes are uncorrelated, whereas higher numbers indicate a higher degree of similarity. The mean similarity score of four independent experiments is shown in ( d ) ( n =4; S.E.M.; * P <0.05)

Article Snippet: The mouse monoclonal anti-FLAG antibody (clone M2, 0.2 μ g/ml) and cytochalasin D were from Sigma-Aldrich (Taufkirchen, Germany); cofilin antibodies were produced in our laboratory; HSC70 antiserum was from Santa Cruz (Heidelberg, Germany); the CD95 antibody was a kind gift of PH Krammer (DKFZ, Heidelberg, Germany); Z-VAD-fmk was from Promega (Mannheim, Germany); 7-AAD, Annexin V and anti-active caspase-3 were from BD Bioscience (Heidelberg, Germany); Hoechst 33342, MitoTracker Deep Red FM and DilC 1 (5), as well as TMRE were obtained from Invitrogen (Karlsruhe, Germany); and PJ-34 was purchased from Sigma.

Techniques: Incubation, Staining, Microscopy, Fluorescence, Electron Microscopy, Labeling, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

doi: 10.1038/cddis.2010.36

Figure Lengend Snippet: Cofilin binds to HSC70 under oxidative stress conditions. ( a ) FLAG-tagged wt- or G39-cofilin was expressed in and then precipitated from Jurkat T cells. Lysates were subjected to PAGE and stained with Coomassie blue. The heavy (50 kDa) and light (ca. 25 kDa) chains of the precipitating antibody were found in each lane, including the control (untransfected Jurkat T cells). Bands that were found only in lane 2 (G39-cofilin) or lane 3 (wt-cofilin) were analyzed using mass spectrometry and identified as HSC70 (only present in lane 2) or actin (lanes 2 and 3). ( b ) Wt-, G39- or G80-cofilin was precipitated from Jurkat T cells. The corresponding western blot was stained for the FLAG-tag (lower part) or HSC70 (upper part). ( c , d ) PBTs were treated with 50 μ M H 2 O 2 for 7 or 24 h. Thereafter, cells were lysed and endogenous cofilin was immunoprecipitated. The precipitates were subjected to western blot analysis and stained for HSC70 (upper panel) or cofilin (lower panel). The graph in ( d ) shows a quantification of the HSC70 to cofilin ratios of three independent experiments ( n =3; S.E.M.; * P <0.05). ( e ) The subcellular localization of HSC70 in long-term oxidatively stressed (lower part) or control T cells (upper part) was analyzed using confocal laser scan microscopy. The white color in the merge of the stressed cells shows the colocalization of cofilin and HSC70 with the mitochondria. The figure is representative of three experiments. ( f ) The colocalization of cofilin and mitochondria (upper graph), cofilin and HSC70 (lower graph) or HSC70 and mitochondria (central graph) was evaluated by the calculation of a similarity score of the corresponding probes ( n =3; S.E.M.; * P <0.05)

Article Snippet: The mouse monoclonal anti-FLAG antibody (clone M2, 0.2 μ g/ml) and cytochalasin D were from Sigma-Aldrich (Taufkirchen, Germany); cofilin antibodies were produced in our laboratory; HSC70 antiserum was from Santa Cruz (Heidelberg, Germany); the CD95 antibody was a kind gift of PH Krammer (DKFZ, Heidelberg, Germany); Z-VAD-fmk was from Promega (Mannheim, Germany); 7-AAD, Annexin V and anti-active caspase-3 were from BD Bioscience (Heidelberg, Germany); Hoechst 33342, MitoTracker Deep Red FM and DilC 1 (5), as well as TMRE were obtained from Invitrogen (Karlsruhe, Germany); and PJ-34 was purchased from Sigma.

Techniques: Staining, Control, Mass Spectrometry, Western Blot, FLAG-tag, Immunoprecipitation, Microscopy

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 1. Decreased myelination was observed in mouse models for the study of depression. (A and B) Representative immunofluorescence images of MBP expression in brain sections from control (A) and CUMS (B) mice. Panels on the right are higher-magnification images of the ventral hippocampus (vHip) and external capsule (ec). Scale bar: 1 mm; original magnification, ×100 (enlarged insets). (C) Quantification of MBP fluorescence intensity in the ventral hippocampus (t10 = 5.681) and the external capsule (t10 = 6.130). n = 6 slices from 3 animal brains/group. (D and E) Representative images of LFB histological staining. Scale bar: 1 mm; original magnification, ×200 (enlarged insets). (F) Quantification results of LFB staining of the ventral hippocampus (t22 = 4.410) and the external capsule (t22 = 12.40). n = 12 slices from 4 animal brains/group. (G–J) Western blots and analysis showing lower MBP expression in ventral hippocampus from CUMS mice (G and H) (t10 = 2.446, n = 6 brains/group) and LPS-treated mice (I and J) (t10 = 2.291, n = 6 brains/group) mice. β-Actin was used as the loading control. Data are shown as the mean ± SEM. *P < 0.05 and ***P < 0.001, by unpaired Student’s t test (C, F, H, and J).

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Immunofluorescence, Expressing, Control, Fluorescence, Staining, Western Blot

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 2. Demyelination and altered synaptic protein expression are observed in mouse models relevant to depression. (A) Schematic diagram of the myelin sheath, showing the nodes of Ranvier, the paranode, and the juxtaparanode, with Caspr 1 expressed mainly in the paranode. (B) Representative images showing Caspr-positive, red-stained paranodal regions in the ventral hippocampus. Scale bar: 20 μm. n = 4 mice/group. (C) High-magnification images of Caspr staining from B. Original magnification, ×400. (D) Nodal lengths were increased in CUMS mice, based on measurements of Caspr-stained regions (n = 50 nodes from 3 different mice/group). (E) Histograms showing the frequency distribution of nodal length, which differed between control and CUMS mice. (F) Representative electron microscopic images showing demyelination in CUMS mice. Scale bar: 500 nm. (G) Thinner myelin sheaths were observed in CUMS mice, as measured by electron microscopy. The total number of myelin sheaths analyzed in the control and CUMS groups was 73 and 87, respectively (n = 15 images from 5 mice/group, t158 = 3.361). (H and I) Representative blots showing decreased PSD95 protein expression in CUMS mice and results of the densitometric analysis. Na+K+ATPase was used as the loading control (n = 6 mice/group, t10 = 3.798). (J and K) Representative blots showing decreased PSD95 protein expression in LPS-treated mice and results of the densitometric analysis (n = 6 mice/group, t10 = 3.866). Data are shown as the mean ± SEM. **P < 0.01 and ***P < 0.001, by unpaired Student’s t test (D, G, I, and K).

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Expressing, Staining, Control, Electron Microscopy

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 3. Clemastine promotes myelination and rescues depression-related behaviors in mice. (A) Schematic outline of clemastine treatment experi- ment in CUMS mice. (B–D) Behavioral testing of CUMS mice and clemastine treatment: (B) SPT [F (2, 34) = 4.657, CUMS plus vehicle: n = 12 mice; CUMS plus clemastine: n = 14 mice; control plus vehicle: n = 11 mice]; (C) OFT [F (2, 39) = 4.843]; and (D) TST [F (2, 39) = 5.197, CUMS plus vehicle: n = 15 mice; CUMS plus clemastine: n = 15 mice; control plus vehicle: n = 12 mice in the OFT and TST]. (E) Schematic outline of clemastine treatment experiment in LPS-treated mice. (F–H) Behavioral testing of LPS-treated mice and clemastine treatment: (F) SPT [F (2, 33) = 8.388]; (G) OFT [F (2, 33) = 12.13]; and (H) TST [F (2, 33) = 5.023] (n = 12 mice/group). (I and J) Western blots and analysis showing lower levels of MBP that were restored by clemastine treatment in CUMS mice [n = 3 brains/group, F (2, 6) = 7.113]. (K and L) Western blots and analysis showing that clemastine treatment restored the diminished expres- sion of MBP caused by LPS [n = 4–5 brains/group, F (2, 10) = 6.098]. (M) Representative electron microscopic images of ventral hippocampus myelinated axons from CUMS mice treated with clemastine and from control groups (n = 8 images from 3 mice/group). Scale bar: 500 nm. (N) Clemastine restored decreased myelin sheath thickness in CUMS mice, based on measurements from electron microscopic images [F (2, 85) = 18.81]. (O) The g-ratio of the inner to outer diameter of myelin sheaths plotted against the axon diameter. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Dunnett’s post hoc comparison.

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Control, Western Blot, Comparison

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 4. Clemastine reverses synaptic deficits. (A) Representative electron microscopic images of CUMS mice showing synaptic deficits that were rescued by clemastine (n = 3 mice/group). Scale bar: 500 nm. (B) The reduction in asymmetric synapses resulting from CUMS was rescued by clemastine [F (2, 16) = 6.063]. (C) Frequency distributions of PSD thickness. (D) Clemastine treatment normalized PSD thickness in CUMS mice to control levels [n = 40 asymmetric synapses from 3 mice in control and vehicle-treated groups; n = 42 asymmetric synapses from 3 mice in CUMS plus the vehicle group; n = 43 asymmetric synapses from 3 mice in the CUMS plus the clemastine-treated group, F (2, 122) = 14.50]. Data are shown as the mean ± SEM. *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Dunnett’s post hoc comparison test.

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 5. EphA4 knockdown rescues CUMS-induced depression-related phenotypes in mice. (A) Volcano plot of DEGs in CUMS mice versus controls. Cutoff values for the adjusted P value and fold change were set at 0.05 and 1.5, respectively. (B and C) Western blot and analysis showing increased EphA4 in hippocampus after CUMS (n = 6 mice/group, t10 = 2.756). (D and E) Western blot and analysis showing increased EphA4 in hippocampus after LPS injection (n = 6 mice/group, t10 = 3.080). (F and G) The level of ubiquitinated EphA4 was dramatically decreased in CUMS mice (n = 5–6 mice/group, t9 = 6.918). (H) Diagram outlining the layout of the AAV shRNA vector used to knock down EphA4 and the experimental timeline. (I and K) Behavioral effects of EphA4 knockdown in the (I) SPT [F (2, 30) = 8.580]; (J) OFT [F (2, 26) = 4.712; and (K) TST [F (2, 30) = 4.961] in CUMS mice (n = 9–11 mice/group). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Dunnett’s post hoc comparisons test (I–K) and unpaired Student’s t test (C, E, and G).

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Knockdown, Western Blot, Injection, shRNA, Plasmid Preparation

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 6. EphA4 knockdown in mice rescues synaptic deficits caused by CUMS. (A and B) Western blot analysis showing lower levels of MBP in CUMS mice restored by EphA4 knockdown [n = 3 brains/group, F (2, 6) = 7.264]. (C) Representative Western blot images of PSD95 protein levels; Na+K+ATPase was used as the protein loading control. (D) Densitometric analysis of PSD95 levels shows that EphA4 knockdown restored the decrease caused by CUMS versus control levels [n = 3 brains/group, F (2, 6) = 8.407]. (E) Representative electron microscopic images of ultrastructure of synapses from the 3 treatment groups. Scale bar: 1.0 μm. (F) Quantification of asymmetric synapse density, showing that EphA4 rescued the decrease caused by CUMS [n = 11 images from 3 mice/group, F (2, 30) = 7.500]. (G) Histograms showing the differential distribution patterns of the PSD thickness. (H) EphA4 knockdown restored the reduced PSD thickness caused by CUMS [n = 61 asymmetric synapses analyzed from 3 mice in the control plus the shNC group, n = 62 asym- metric synapses from 3 mice in CUMS plus the shNC and CUMS plus shEpha4 groups, F (2, 182) = 21.79]. Data are shown are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA with Dunnett’s post hoc comparison (B, D, F, and H). ctrl, control.

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Knockdown, Western Blot, Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: The Eph receptor A4 plays a role in demyelination and depression-related behavior

doi: 10.1172/jci152187

Figure Lengend Snippet: Figure 8. Specific knockdown of EphA4 expression in excitatory neurons in mice can inhibit demyelination and rescue the synaptic deficits induced by CUMS. (A and B) Lower levels of MBP in CUMS mice restored by EphA4 knockdown in excitatory neurons [n = 4–5 brains/group, F (2, 10) = 11.51]. (C) Repre- sentative electron microscopic images of myelinated axons. Scale bar: 500 nm. (D) Specific knockdown of EphA4 in excitatory neurons restored decreased myelin sheath thickness in CUMS mice [n = 85 myelinated axons from 3 mice/group, (F (2, 252) = 166.1]. (E) Representative electron microscopic images of synapses from 3 treatment groups. Scale bar: 500 nm. (F and G) EphA4 knockdown in excitatory neurons restored the reduced asymmetric synapse numbers (F) [F (2, 20) = 14.32] and PSD thickness (G) [n = 85 asymmetric synapses analyzed from 3 mice/group, F (2, 252) = 44.57] caused by CUMS. Data are shown as the mean ± SEM. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with post hoc comparisons with Dunnett’s test.

Article Snippet: Identification of EphA4-interacting proteins by mass spectrometry combined with immunoprecipitation To identify EphA4-interacting proteins, protein lysate (total protein amount: 600–700 μg) from ventral hippocampus of control and CUMS mice was incubated with EphA4 antibody (Santa Cruz Biotechnology, sc-365503, 1:50) or normal mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:100) in a rotator at 4°C for 16–18 hours.

Techniques: Knockdown, Expressing

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 1. SIAH-1 and -2 promote -synuclein monoubiquitylation in vitro. A, His--synuclein was incubated with recombinant SIAH-1 or SIAH-2, UbcH5b, ubiquitin, and the other purified components of the ubiquitin (Ubiq) system.LevelsofHis--synucleinubiquitylationweredeterminedbyWestern blot using an anti--synuclein antibody. B, -synuclein monoubiquitylation revealed by conjugation with lysine-less ubiquitin (ubiquitin K0) and methy- lated ubiquitin (MetUbiq). His--synuclein was incubated with recombinant SIAH-2, UbcH5b, and the purified components of the ubiquitin system, in the presence of ubiquitin, ubiquitin K0, or methylated ubiquitin. Levels of His-- synuclein ubiquitylation were determined by Western blot using an antibody to -synuclein. C, -synuclein is specifically monoubiquitylated by SIAH-2. His--synuclein was incubated with UbcH5b or UbcH7, ubiquitin K0, and the indicated components of the ubiquitin systems, in the presence of different recombinant E3 ubiquitin-ligases. The levels of His--synuclein ubiquityla- tion were determined by Western blot using an anti--synuclein antibody. The figure panels are representative of 3–4 independent experiments. D, His--synuclein, SIAH-2, UbcH5b, ubiquitin, and the purified components of the ubiquitin system were incubated at 37 °C to obtain in vitro monoubiq- uitylated -synuclein. Monoubiquitylated -synuclein was run in a SDS-PAGE and visualized by Coomassie Blue staining. Indicated monoubiquitylated -synuclein was sent to mass spectrometry analysis. E1, ubiquitin-activating enzyme.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: In Vitro, Incubation, Recombinant, Ubiquitin Proteomics, Purification, Conjugation Assay, Methylation, Western Blot, SDS Page, Staining, Mass Spectrometry

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 2. Mutation of -synuclein targeted lysines. His--synuclein (wild- type (WT), K96R or K6R,K10R,K12R,K21R,K23R,K32R,K34R,K43R,K45R,K96R (K) mutant) was incubated with recombinant SIAH-2, UbcH5b, ubiquitin, and the other purified components of the ubiquitin system as described under “Experimental Procedures.” Levels of His--synuclein ubiquitylation were determined by Western blot using a polyclonal anti--synuclein antibody.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Mutagenesis, Incubation, Recombinant, Ubiquitin Proteomics, Purification, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 3. SIAH monoubiquitylates in vivo and co-immunoprecipitates with -synuclein from brain tis- sue. A, effect of proteolytic inhibitors on -synuclein steady-state levels. SH-SY5Y cells were transfected with HA--synuclein and incubated 12 h with 10 M lactacystin, 10 mM NH4Cl, 10 mM 3-MA, or a combination of all three inhibitors. HA--synuclein from total cell lysates was detected by Western blot using an antibody to -synuclein. Loading control was monitored with anti-actin antibody (lower panel). B, SIAH-2 overexpression increases -synuclein monoubiquitylation. SH-SY5Y cells were transfected with HA--synuclein, FLAG-ubiq- uitin, in the absence or in the presence of myc-SIAH-2. Cells were incubated 12 h with 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. HA--synuclein was immunoprecipitated (IP) with an anti-HA antibody, and monou- biquitylated -synuclein was detected by Western blot using an antibody to -synuclein. The middle panel shows the levels of immunoprecipitated -synuclein under short exposure of the blot. The lower panel shows the levels of SIAH-2 by Western blot using an anti-Myc antibody. C, SIAH-1 and -2 interact in vivo with -synuclein. SIAH-1 and -2 were immunoprecipitated from rat brain lysate using goat anti-SIAH-1/2 antibody, and detection of co-immunoprecipitation was carried out using rabbit anti--synuclein antibody. -Synuclein wasdetectedinSIAH-immunoprecipitatebutnotinbeadsalone.D,SIAH-1wasimmunoprecipitatedwithfrom rat brain lysate using goat anti-SIAH-1 antibody, and detection of co-immunoprecipitation was carried out using rabbit anti--synuclein antibody. -Synuclein was detected in SIAH-1-immunoprecipitate but not in beads alone or p150glued-immunoprecipitate.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: In Vivo, Transfection, Incubation, Western Blot, Control, Over Expression, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 4. Endogenous SIAH monoubiquitylates -synuclein. A, SH-SY5Y cells were transfected with HA-- synuclein, FLAG-ubiquitin, in the absence or presence of shRNA to SIAH-1 and SIAH-2. Cells were incubated 12 h with 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. HA--synuclein was immunoprecipitated (IP) with an anti-HA antibody, and monoubiquitylated -synuclein was detected by Western blot using an anti-- synuclein antibody. The lower panel shows the suppression of SIAH-1 and -2 expressions by the shRNAs using anti-SIAH-1 and -2 antibodies. B, monoubiquitylated -synuclein is among the pool of ubiquitylated proteins. SH-SY5Y cells were transfected with HA--synuclein, FLAG-ubiquitin, in the absence or presence of either myc-SIAH-2 or shRNAs to SIAH-1 and SIAH-2. Cells were incubated 12 h with 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. FLAG-ubiquitylated proteins were immunoprecipitated with an anti-FLAG antibody, and monoubiquitylated -synuclein was detected by Western blot using an antibody to -synuclein. The figure panels are representative of three independent experiments.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Transfection, Ubiquitin Proteomics, shRNA, Incubation, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 5. Deubiquitinasesanddifferentproteolyticpathwaysmodulatetheamountofmonoubiquitylated -synuclein.A,SH-SY5YcellsweretransfectedwithHA--synuclein,myc-SIAH-2,inthepresenceofFLAG-ubiquitin, FLAG-ubiquitinK0,orFLAG-ubiquitinG76Amutant.Cellswereincubated12hwith10Mlactacystin,10mMNH4Cl, and 10 mM 3-MA. HA--synuclein was immunoprecipitated (IP) with an anti-HA antibody and ubiquitylated -synuclein was detected by Western blot using an anti--synuclein antibody. The lower panel shows the levels of SIAH-2 by Western blot using an anti-Myc antibody. B, SH-SY5Y cells were transfected with HA--synuclein, myc- SIAH-2,andFLAG-ubiquitinG76Amutant.Cellswereincubated12hwithMe2SOasvehicle,10Mlactacystin,10mM NH4Cl, 10 mM 3-MA or all the three compounds combined. HA--synuclein was immunoprecipitated with an anti-HA antibody and monoubiquitylated -synuclein was detected by Western blot using an antibody to -synuclein.ThelowerpanelshowsthelevelsofSIAH-2byWesternblotusingananti-Mycantibody.Thefiguresare representative of three independent experiments. WT, wild type.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE6.Monoubiquitylationof-synucleinbySIAHincreasesitsaggregationinvitro.A,His--synuclein was incubated with UbcH5b and the indicated components of the ubiquitin (Ubiq) system, in the absence or in the presence of recombinant SIAH-2. His--synuclein was then incubated at 65 °C for additional 10 h to pro- mote its aggregation. His--synuclein ubiquitylation and aggregation were determined by Western blot using an antibody to -synuclein. Asterisk indicates the position of -synuclein dimer. B, His--synuclein was incu- bated with UbcH5b and indicated components of the ubiquitin system, in the absence or in the presence of recombinantSIAH-2.His--synucleinwasincubatedat65 °Cfor2htoallowitsaggregationandthenseparated into soluble (Sol) and insoluble (Insol) fractions by 16,000 g centrifugation. The levels of His--synuclein monoubiquitylation and aggregation were determined by Western blot using an anti--synuclein antibody. The figures are representative of four independent experiments. C, electron micrographs showing increased -synuclein aggregation by ubiquitylation. His--synuclein was incubated with recombinant SIAH-2, compo- nents of the ubiquitin system, in the absence (left panel) or in the presence of UbcH5b (middle and right panels). Reaction mixtures were then incubated at 65 °C for additional 16 h to promote -synuclein aggregation. His--synuclein aggregation was determined by transmission electron microscopy analysis. Middle and right panels show the aggregation of -synuclein monoubiquitylated at 30 and 10%, using 0.1 and 0.3 g of SIAH, respectively. Magnification was 15,000 times. The figures are representative of three independent experi- ments. D, titration of -synuclein monoubiquitylation by SIAH-2. His--synuclein was incubated with increas- ing amounts of SIAH-2, UbcH5b, and purified components of the ubiquitin system. Levels of His--synuclein ubiquitylation were determined by Western blot using anti--synuclein antibody. E, densitometric quantifica- tion of the -synuclein monoubiquitylation levels shown in D. Error bars represent standard error of four independent experiments. Highlighted condition in D and E represents 10% of monoubiquitylated -synuclein. MetUbiq, methylated ubiquitin; E1, ubiquitin-activating enzyme.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Incubation, Ubiquitin Proteomics, Recombinant, Western Blot, Centrifugation, Transmission Assay, Electron Microscopy, Titration, Purification, Methylation

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 7. Monoubiquitylation of -synuclein by SIAH enhances its inclusion formation in human dopa- minergic cells. A, SH-SY5Y cells were transfected with HA--synuclein and myc-SIAH-2, and incubated for 12 h with Me2SO (DMSO) as vehicle or a combination of 10 M lactacystin (Lacta), 10 mM NH4Cl, and 10 mM 3-MA. Immunocytochemistrywascarriedoutusinganti-HA(red)andanti-Myc(green)antibodies.Nucleiwerestained with TOPRO-3. Scale bar, 25 m. Arrows point to -synuclein/SIAH inclusions, and an arrowhead points to SIAH inclusion devoid of -synuclein. B, SH-SY5Y cells were transfected with HA--synuclein, in the absence and presence of shRNAs to SIAH-1 and SIAH-2, and incubated for 12 h with a combination of 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. Immunocytochemistry was carried out using anti-HA (red) antibody. Scale bar, 25 m. C, quantification of the percent of inclusion body formation in SH-SY5Y cells. The cells were transfected with HA--synuclein with vector alone, shRNAs to SIAH-1 and -2, or myc-SIAH-2. The cells were treated for 12 h with Me2SO as vehicle, 10 M lactacystin or a combination of 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. Error bars represent standard error of 3–8 independent experiments. **, significantly different from vehicle control at p 0.01. a, significantly different from the values observed with vector alone or shRNAs to SIAH-1 and -2 at p 0.01.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Transfection, Incubation, Immunocytochemistry, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 9. Characterization of -synuclein inclusions formed by SIAH. A, -synuclein (-Syn) inclusions are thioflavin (Thio)-positive. SH-SY5Y cells were transfected with HA--synuclein and myc-SIAH-2 and incubated for 12 h with a combination of 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. Immunocytochemistry was carried out using anti-HA antibody (red) and thio- flavin S staining (green). Scale bar, 25 m. B, -synuclein inclusions contain additional PD-related proteins. SH-SY5Y cells were transfected with HA-- synuclein and untagged SIAH-2, with FLAG-ubiquitin (Ubiq), myc-synphilin-1, or myc-UCH-L1. Cells were incubated for 12 h with a combination of 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA, and immunocytochemistry was carried out using anti-HA (red) and anti-FLAG or anti-Myc antibodies (green). Scale bar, 25 m. The figures are representative of three independent experiments.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Transfection, Incubation, Immunocytochemistry, Staining, Ubiquitin Proteomics

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 10. Monoubiquitylation of -synuclein A53T mutant increases its tendency to form inclusions in human dopaminergic cells. A, His-- synuclein mutants (wild-type (WT), A53T, A30P, or E46K) were incubated with recombinant SIAH-2, UbcH5b, ubiquitin K0, and the indicated components of the ubiquitin system. The levels of His--synuclein ubiquitylation were deter- mined by Western blot using an antibody to -synuclein (-Syn). B, SH-SY5Y cells were transfected with HA--synuclein (wild-type or A53T mutant) and incubated with 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA for 12 h. Immunocytochemistry was carried out using anti-HA (red) and anti-Myc (green) antibodies. Nuclei were stained with TOPRO-3. Scale bar, 25 m. C, quantification of the percent of inclusion body formation in SH-SY5Y cells transfected with HA--synuclein (wild-type, A53T, or A30P mutant) and incu- bated with Me2SO as vehicle or 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA for 12 h. Error bars represent standard error of 3–4 independent exper- iments. **, significantly different from Me2SO vehicle control at p 0.01. a, significantly different from the values of wild-type at p 0.05.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Mutagenesis, Incubation, Recombinant, Ubiquitin Proteomics, Western Blot, Transfection, Immunocytochemistry, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

doi: 10.1074/jbc.m704809200

Figure Lengend Snippet: FIGURE 11. Monoubiquitylated -synuclein inclusions are toxic to SH-SY5Y cells. Shown is the quantification of the percentage of cell death in SH-SY5Y cells transfected with HA--synuclein and myc-SIAH-2 and incu- bated for 12 h with a combination of 10 M lactacystin, 10 mM NH4Cl, and 10 mM 3-MA. The graph depicts the amount of cell death in cells lacking (empty bar)orcontaining(filledbar)-synucleininclusions.***,significantlydifferent from control at p 0.001.

Article Snippet: Brain homogenates were clarified by centrifugation at 13,000 g for 5 min. Antibodies to SIAH-1/2 or SIAH-1 (H-18 and N-15, respectively) (Santa Cruz Biotechnology) were coupled to protein G beads (22) and incubated for 7 h with brain homogenate (2 mg/ml).

Techniques: Transfection, Control

Journal: Chemical Engineering Journal

Article Title: Establishment of human minor salivary gland organoids in laminin-GelMA hydrogel from healthy individuals and Sjögren’s disease patients

doi: 10.1016/j.cej.2024.158257

Figure Lengend Snippet: Fig. 2. Artificial laminin-GelMA hydrogel characteristics and the formation of hMSG organoids in this hydrogel. A. Schematic illustration showing hMSG encap sulation in the artificial laminin-GelMA hydrogel.B. Photographs depicting the sol-gel process of GelMA and laminin-GelMA hydrogels under UV irradiation.C. Ultrastructure of Matrigel, GelMA, and laminin-GelMA hydrogels obtained by SEM (scale bar = 100 μm).D. Methacrylamide signals of crosslinked laminin-GelMA in an NMR hydrogen spectrum.E. Storage modulus and compression test results of laminin-GelMA hydrogel.F. Immunofluorescence staining of hMSG tissue (top) and hMSG organoids (bottom) cultured in Matrigel for laminin (green) and KI67 (red). Nuclei are stained with DAPI (blue) (scale bar = 100 μm).G. hMSG organoid maintained in laminin-GelMA hydrogel and tracked in a series of time-lapse images (scale bar = 500 μm).H. Immunofluorescence staining of hMSG organoids (cultured in laminin-GelMA hydrogel) for E-cadherin (green)/AQP5 (red), CK5 (green)/CK7 (red), MUC5B (green)/AMY1 (red), and PANCK (green)/αSMA (red). Nuclei stained with DAPI (blue) (scale bar = 100 μm).I. mRNA expression of ductal (CK5 and CK7), acinar (AQP5 and MIST1), myoepithelial (CK14 and ACTA2), proliferation (KI67), stemness (SOX2, SOX9, and SOX10), and amylase (AMY1) markers in hMSG organoids cultured in GelMA and laminin-GelMA hydrogels.J. Whole-exome sequencing heatmap showing differentially expressed genes in hMSG organoids grown in laminin-GelMA hydrogel. K. Immunofluorescence staining of hMSG organoids (cultured in laminin-GelMA hydrogel) for Integrin α3(red) and Integrin β3 (green). Nuclei stained with DAPI (blue) (scale bar = 20 μm).(hMSG: human minor salivary gland; GelMA: Gelatin Methacryloyl; LAP: lithium phenyl-2,4,6-trimethylbenzoylphosphinate; SEM: Scanning Electron Microscopy; NMR: Nuclear magnetic resonance).

Article Snippet: The primary antibodies and corresponding TSA used for each protein were as follows: anti-SMA (67735, Proteintech) and Opal 520 for SMA; anti-human nucleoli (ab190710, Abcam) and Opal 540 for human nucleoli; anti-CK5 (WH109006, ABclonal) and Opal 570 for CK5; antiAQP5 (ab92320, Abcam) and Opal 620 for AQP5; anti-KI67 (ab1667, Abcam) and Opal 650 for KI67; and anti-CK7 (66483, Proteintech) and Opal 690 for CK7.

Techniques: Irradiation, Immunofluorescence, Staining, Cell Culture, Expressing, Sequencing, Electron Microscopy, Nuclear Magnetic Resonance

Journal: Chemical Engineering Journal

Article Title: Establishment of human minor salivary gland organoids in laminin-GelMA hydrogel from healthy individuals and Sjögren’s disease patients

doi: 10.1016/j.cej.2024.158257

Figure Lengend Snippet: Fig. 4. Orthotopic transplantation of hMSG organoids and assessment of their regenerative potential. A. Schematic illustration of hMSG organoids transplanted into partially defective submandibular glands (SMG) of NSG mice. B. Image of hMSG organoids expressing green fluorescent protein (GFP) (scale bar = 1 mm). C. The GFP-expressing transplant was observed using two-photon laser confocal microscopy (scale bar = 100 μm). D. In vivo fluorescent signal imaging of hMSG- transplanted NSG mice after 2 months. E. Immunohistochemistry staining of human nucleoli in murine SMG transplanted with hMSG organoids (scale bar = 50 μm). F. Multiplexed immunofluorescence staining of murine SMG transplanted with hMSGs organoids for CK7, KI67, AQP5, CK5, ACTA2, and human nucleoli. Nuclei stained with DAPI (blue) (scale bar = 50 μm). G. Venn diagram showing overlap between human and murine salivary proteins in the collected saliva from hMSG- transplanted murine glands. H. Mass spectrometry images of human-specific peptide fragments of AMY1A detected in the collected saliva from hMSG-transplanted murine glands.

Article Snippet: The primary antibodies and corresponding TSA used for each protein were as follows: anti-SMA (67735, Proteintech) and Opal 520 for SMA; anti-human nucleoli (ab190710, Abcam) and Opal 540 for human nucleoli; anti-CK5 (WH109006, ABclonal) and Opal 570 for CK5; antiAQP5 (ab92320, Abcam) and Opal 620 for AQP5; anti-KI67 (ab1667, Abcam) and Opal 650 for KI67; and anti-CK7 (66483, Proteintech) and Opal 690 for CK7.

Techniques: Transplantation Assay, Expressing, Confocal Microscopy, In Vivo, Imaging, Immunohistochemistry, Staining, Immunofluorescence, Mass Spectrometry

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 1. Downregulation of NLRX1 correlates with aggravated human NP cell senescence and IDD progression. Human NP tissue specimens with different degenerative grades were collected for histological analysis. (A) Representative MRI images at T2 weight sequence were evaluated by Pfirrmann grading system. II: grade II, III: grade III, IV: grade IV. (B) histological analysis of human NP samples by alcian blue staining, scale bar: 100 μm. (C) immunohistochemical staining of CDKN2A, MKI67 and NLRX1 in different degenerative NP tissues, scale bar: 100 μm. (D and E) linear regression analyses of the tissue staining intensity of CDKN2A and that of NLRX1 (D), or the intensity of MKI67 and that of NLRX1 (E). AOD, average optical density. (F-H) protein expressions of senescence indicators (TP53, CDKN1A, CDKN2A), SASP factors (IL1B, IL6) and NLRX1 in primary human NP cells isolated from different degenerative NP tissues with the treatment of TBHP (100 μM), as determined by western blotting. (I-L) cell senescence (SA-GLB1/β-gal staining), cell proliferation (EdU incorporation) and NLRX1 expression (immunofluorescent staining) in primary human NP cells isolated from different degenerative NP tissues with the treatment of TBHP (100 μM), scale bar: 100 μm. (M and N) MRI examination, hematoxylin and eosin (HE) and safranin-O (SO) staining in sham or operation-induced degenerated disc of rat, scale bar: 500 μm (left panel), 50 μm (right panel). (O and P) immunohistochemical staining of aggrecan, collagen type II, NLRX1 and CDKN2A in sham or operation-induced degenerated disc of rat, scale bar: 500 μm (left panel), 50 μm (right panel). Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Sequencing, Staining, Immunohistochemical staining, Isolation, Western Blot, Expressing

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 2. NLRX1 promotes NP cell proliferation, attenuates cell senescence and senescence-associated secretory phenotypes (SASPs) and rescues disc degeneration. Primary human NP cells isolated from NP tissues with different Pfirrmann grades were prepared. (A) knockdown efficiency of NLRX1 in NP cells by siRNA, analyzed with western blotting and RT-qPCR. (B-D) cell proliferation (EdU incorporation) and cell senescence (SA-GLB1/β-gal staining) in primary human NP cells isolated from health NP tissues treated by PBS or TBHP with NLRX1 knockdown or not, scale bar: 100 μm. (E and F) protein expressions of senescence indicators (TP53, CDKN1A, CDKN2A), SASP factors (IL1B) in primary human NP cells isolated from health NP tissues treated by PBS or TBHP with NLRX1 knockdown or not, as determined by western blotting. (G and H) protein expressions of senescence indicators (TP53, CDKN1A, CDKN2A), SASP factors (IL1B) and cell proliferation (MKI67) in primary human NP cells isolated from degenerated NP tissues with NLRX1 overexpression or not, as determined by western blotting. (I and J) cell proliferation (EdU incorporation) and cell senescence (SA-GLB1/β-gal staining) in primary human NP cells isolated from degenerated NP tissues with NLRX1 overexpression or not, scale bar: 100 μm. (K) rat disc degenerative models treated with NLRX1 overexpression and histologic analysis, upper panel: HE and so staining, lower panel: immunohistochemical staining of collagen type II, CDKN2A and MKI67, scale bar: 500 μm (left panel), 50 μm (right panel). Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Isolation, Knockdown, Western Blot, Quantitative RT-PCR, Staining, Over Expression, Immunohistochemical staining

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 3. NLRX1 is essential for the beneficial action of mitophagy in alleviating mitochondrial dysfunction and NP cell senescence. Primary human NP cells isolated from NP tissues with different Pfirrmann grades were prepared. (A) confocal analysis of NLRX1 and MAP1LC3B with if staining in NP cells isolated from health NP tissues treated by PBS or TBHP, scale bar: 10 μm. (B) proteins immunoprecipitated (IP) from NP cells isolated from health NP tissues treated by PBS or TBHP followed by western blotting, left panel: IP with anti-MAP1LC3B antibody, right panel: IP with anti-NLRX1 antibody. (C) confocal analysis of TOMM20 and MAP1LC3B with if staining in NP cells isolated from degenerated NP tissues treated by PBS or TBHP with NLRX1 overexpression or not, scale bar: 10 μm. (D and E) protein expressions of mitophagy indicators (MAP1LC3B-II, TOMM20, TIMM23) in primary human NP cells isolated from degenerated NP tissues treated by PBS or TBHP with NLRX1 overexpression or not, as determined by western blotting. (F-I) JC-1 incubation for detecting mitochondrial membrane potential (MMP; F, H) and DCFH incubation for detecting reactive oxidative species (ROS; G, I) in NP cells isolated from degenerated NP tissues treated by PBS, TBHP or 3-MA with NLRX1 overexpression or not, as determined by flow cytometry. (J and K) protein expressions of senescence indicators (TP53, CDKN2A) and SASP factors (IL1B, IL6) in NP cells isolated from degenerated NP tissues treated by PBS, TBHP or 3-MA with NLRX1 overexpression or not, as determined by western blotting. (L and M) cell proliferation (MKI67 immunofluorescent staining, EdU incorporation) and cell senescence (SA-GLB1/β-gal staining) in primary human NP cells isolated from degenerated NP tissues treated by PBS, TBHP or 3-MA with NLRX1 overexpression or not, scale bar: 50 μm (IF images), 100 μm (white light images). Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Isolation, Staining, Immunoprecipitation, Western Blot, Over Expression, Incubation, Membrane, Flow Cytometry

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 4. NLRX1 induces selective mitochondrial fission and mitophagy to maintain adaptive mitochondrial morphology. Primary human NP cells isolated from degenerated NP tissues were prepared. (A) Co-localization analysis of mCherry and GFP in live stable mCherry-GFP-MAP1LC3B-expressing NP cells following the treatments of PBS, TBHP or Baf-A1 with NLRX1 overexpression or not, scale bar: 200 μm. (B and C) protein expressions of mitophagy indicators (MAP1LC3B-II, TOMM20, TIMM23) in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS, TBHP or Baf-A1 with NLRX1 overexpression or not, as determined by western blotting. (D-F) mitochondrial morphology analysis by fluorescence microscope with MitoTracker Red CMXRos label (D and E) and transmission electron microscopy (TEM) (F) in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or TBHP with NLRX1 overexpression or not, fluorescent scale bar: 5 μm, TEM scale bar: 2 μm (upper panel), 500 nm (lower panel). (G and H) protein expressions of mitochondrial dynamics indicators (p-DNM1L, DNM1L, MFF, MFN1, MFN2, OPA1, OMA1) in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or TBHP with NLRX1 overexpression or not. (I and J) protein expressions of PINK1 and PRKN in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or TBHP with NLRX1 overexpression or not. (K) confocal analysis of MitoTracker labeling and PRKN protein with if staining in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or TBHP with NLRX1 overexpression or not, scale bar: 10 μm. Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Isolation, Expressing, Over Expression, Western Blot, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Labeling, Staining

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 5. Zinc transporter SLC39A7 interacts with NLRX1 in NP cells. (A) the proteins that potentially interact with NLRX1 in human NP cells were immunopreci pitated with anti-NLRX1 antibody and analyzed by mass spectrometry. (B) endogenous protein immunoprecipitated (IP) from NP cells followed by western blotting, left panel: IP with anti-SLC39A7 antibody, right panel: IP with anti-NLRX1 antibody. (C) exogenous protein immunoprecipitated (IP) from 293T cells with plasmid transfection (mock GFP or HA, GFP-NLRX1, HA-SLC39A7) followed by western blotting, left panel: IP with anti-GFP antibody, right panel: IP with anti-HA antibody. (D) endogenous protein immunoprecipitated (IP) from NP cells isolated from health NP tissues following the treatments of PBS or TBHP, followed by western blotting, left panel: IP with anti-SLC39A7 antibody, right panel: IP with anti-NLRX1 antibody. (E) confocal analysis of NLRX1 and SLC39A7 with if staining in NP cells isolated from health NP tissues treated by PBS or TBHP, scale bar: 10 μm. Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Plasmid Preparation, Transfection, Isolation, Staining

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 7. NLRX1 participates in modulating SLC39A7 trafficking at mitochondria that is essential for selective mitochondrial fission and mitophagy. (A and B) the expression of SLC39A7 in human different degenerative NP tissues (A) and rat disc degenerative models (B) examined by IHC and IF, human tissue scale bar: 25 μm (upper panel), 50 μm (lower panel), rat scale bar: 500 μm (upper panel), 500 μm (lower panel). (C) confocal analysis of MitoTracker labeling and SLC39A7 protein with if staining in primary human NP cells isolated from health NP tissues with NLRX1 knockdown or not (upper two panel), or from degenerated NP tissues with NLRX1 overexpression or not (lower two panel), scale bar: 10 μm. (D and E) protein lysates extracted specially in mitochondria and cytoplasm of human NP cells isolated from health NP tissues with NLRX1 knockdown or not (D), or from degenerated NP tissues with NLRX1 overexpression or not (E), followed by western blotting. (F) protein immunoprecipitated (IP) using anti-NLRX1 antibody from NP cells treated by NX-13 or not, followed by western blotting. (G) protein lysates extracted

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Expressing, Labeling, Staining, Isolation, Knockdown, Over Expression, Western Blot, Immunoprecipitation

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 8. Pharmacological activation of NLRX1 by NX-13 attenuates disc degeneration in rat degenerative disc model and tissue culture of human disc. (A and B) protein expressions of mitophagy indicators (MAP1LC3B-II, TOMM20, TIMM23), senescence indicators (TP53, CDKN2A) and SASP factors (IL1B, IL6) in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or NX-13. (C) confocal analysis of TOMM20 and MAP1LC3B protein with if staining in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or NX-13, scale bar: 10 μm. (D) cell proliferation (MKI67 immunofluorescent staining) and cell senescence (SA-GLB1/β-gal staining) in primary human NP cells isolated from degenerated NP tissues following the treatments of PBS or NX-13, scale bar: 50 μm (IF images), 100 μm (white light images). (E-G) rat disc degenerative models treated with NX-13 and histologic analysis, upper panel: HE and so staining, lower panel: immunohisto chemical staining of collagen type II, CDKN2A and MKI67, scale bar: 500 μm (left panel), 50 μm (right panel). (H and I) ROS detection by DCFH labelling in frozen section of human NP tissues in-vitro cultured by NX-13 or not, scale bar: 500 μm. (J-M) histologic analysis of human NP tissues in-vitro cultured by NX-13 or not with alcian blue staining or IHC staining (MKI67, CDKN2A), scale bar: 20 μm. Data are represented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Activation Assay, Isolation, Staining, In Vitro, Cell Culture, Immunohistochemistry

Journal: Autophagy

Article Title: The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn 2+ trafficking.

doi: 10.1080/15548627.2023.2274205

Figure Lengend Snippet: Figure 9. Schematic depicting the molecular mechanism through which NLRX1-SLC39A7 facilitates intervertebral disc rejuvenation via orchestrating mitochondrial dynamics and mitophagy. NLRX1 recruits zinc transporter SLC39A7 to mitochondrial membrane in order to maintain mitochondrial zinc homeostasis, that is essential for selective segregation of damaged mitochondria during mitochondrial dynamics and subsequent degradation by mitophagy. The coordinated network of mitochondrial dynamics and mitophagy is essential for mitochondrial homeostasis and disc rejuvenation. Conversely, IDD progression is characterized by decreased expression of NLRX1 in NP cells, which results in SLC39A7 loss and aberrant zinc accumulation in mitochondria. Consequently, excessive mitochondrial fission and mitophagy aggravate NP cell senescence and IDD progression.

Article Snippet: After blocking, the sections were probed with primary antibodies against SLC39A7 (1:100; Affinity, DF4635) at 4°C overnight and incubated with an Alexa Fluor 568- conjugated anti-rabbit secondary antibody at room temperature for 1 h. For cell slide staining, NP cells at 60% confluence were exposed to certain treatments and then fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. After permeabilization and blocking, the cell slides were incubated with primary antibodies against NLRX1 (1:100; Affinity, DF12124), SLC39A7 (1:100; Affinity, DF4635), TOMM20 (1:100; Proteintech 11,802–1-AP), MAP1LC3B (1:100; Affinity, AF4650), and MKI67 (1:100; Affinity, AF0198) and incubated with Alexa Fluor 488-conjguated or Alexa Fluor 568-conjugated anti-rabbit secondary antibodies (Invitrogen, A-11008 for 488 and A-11011 for 568) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Beyotime, P0131-5 ml).

Techniques: Membrane, Expressing

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Characterization of uEVs from healthy controls (A) A schematic of the study outlining the discovery and validation cohort. (B) Negative stain transmission electron microscopy of uEVs. Scale bars, 200 nm. (C) Nanoparticle tracking analysis of uEVs isolated from healthy controls. (D) Venn diagram of total proteins detected in uEVs isolated from healthy controls. (E) Bar plot representing the abundance of common classical exosomal markers (CD63, CD9) and markers for classical microvesicles (Annexin A1) and arrestin-domain-containing protein 1-mediated microvesicles (TSG101) in healthy controls. The yaxis represents log10 relative abundance. (F) KEGG/Wiki pathway analysis (g:profiler) of the 1,298 common proteins in uEVs from healthy controls. The top five terms with the lowest adjusted p values were extracted. (G) Intrarenal expression of top 50 molecules in uEVs from healthy controls visualized using kidney cell explorer ( Ransick et al., 2019 ) in order of relative abundance and immunofluorescence of human kidney specimens of 5 out of 50 molecules. Glutathione hydrolase 1 proenzyme (GGT1), phosphoglycerate kinase 1 (PGK1), uromodulin (UMOD), annexin A11 (ANXA11), keratin, and type I cytoskeletal 14 (KRT14) are expressed in the proximal tubule, loop of Henle, the distal tubule, collecting duct, and deep medullary epithelium of pelvis, respectively. The numbers above the figure represent each nephron segment: 1, podocytes; 2, parietal epithelium; 3, proximal tubule; 4, the loop of Henle; 5, distal tubule; 6, nephron connecting tubule; 7, cortical collecting duct; 8, medullary collecting duct; 9, deep medullary epithelium of pelvis. Scale bars, 200 μm.

Article Snippet: Mouse monoclonal anti-GGT1 antibody (Cat# sc-166908, Santa Cruz), mouse monoclonal anti-THP (UMOD) antibody (Cat# sc-271022, Santa Cruz), rabbit polyclonal anti-PGK1 antibody (Cat# 17811-1-AP, Proteintech), rabbit polyclonal anti-KRT14 antibody (Cat# 10143-1-AP, Proteintech), rabbit polyclonal anti-ANXA11 antibody (Cat# 10479-2-AP, Proteintech) and above mentioned anti-CD9 antibody, anti-MGAM antibody, and anti-MUC1 antibody were used as primary antibodies.

Techniques: Biomarker Discovery, Staining, Transmission Assay, Electron Microscopy, Isolation, Expressing, Immunofluorescence

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-GGT1 antibody (Cat# sc-166908, Santa Cruz), mouse monoclonal anti-THP (UMOD) antibody (Cat# sc-271022, Santa Cruz), rabbit polyclonal anti-PGK1 antibody (Cat# 17811-1-AP, Proteintech), rabbit polyclonal anti-KRT14 antibody (Cat# 10143-1-AP, Proteintech), rabbit polyclonal anti-ANXA11 antibody (Cat# 10479-2-AP, Proteintech) and above mentioned anti-CD9 antibody, anti-MGAM antibody, and anti-MUC1 antibody were used as primary antibodies.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy