Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: PLAT-E cells were transfected with retroviral pMX-Flag-NEMO- WT -RFP (NM-WT) and pMX-flag-NEMO- K270 -RFP (NM-KA) expression vector. ( A ) Fluorescence images showing distribution of NM-WT-RFP in cytoplasm compared to puncta (yellow arrows) (juxtaposed to nuclei- DAPI stained) formation in case of NM-KA-RFP in PLAT-E cells. ( B ) Western blot for LC3 using WES (protein simple). BMMs were cultured for 2 days with RANKL (preOC) followed by 6 hr of serum starvation and western blotting. Fold change of LC3 relative to actin is indicated on top. ( C ) Quantification of LC3+ cells per high magnification field. ( D ) For flow cytometry, BMMs were transduced with pMX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells, and flow analysis was done to detect GFP signal or LC3 flux. Contour plots showing LC3-GFP+ expressing cells in NM-WT and NM-KA preOC (Blue: NM-WT without serum starvation, Red: NM-KA without serum starvation, and Black: after 6 hr of serum starvation), ( E ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy (Red histogram: background signal in uninfected cells, Blue histogram: No serum starvation or 10% FBS control, yellow: 6 hr serum starvation, and pink: chloroquine), ( F ) Change in Mean fluorescent intensity (MFI) showing LC3-GFP signal in NM-WT and NM-KA preOC cells post autophagy induction. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05). (*p<0.05, **p<0.01 and ***p<0.001). Figure 4—source data 1. Western blot for LC3 using WES (protein simple). Figure 4—source data 2. Quantification of LC3+ cells. Figure 4—source data 3. LC3-GFP FACS analysis.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transfection, Retroviral, Expressing, Plasmid Preparation, Fluorescence, Staining, Western Blot, Cell Culture, Flow Cytometry, Transduction, Generated, Control

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Pre-OC (RANKL-treated BMMs) from NM-WT and NM-KA mice were pelleted and processed for electron microscopic and Immunofluorescence (IF) analysis after 6 hr of serum starvation. Representative Electron microscopic images (x7500) showing nucleus, Cell membrane (CM) lysosome ( L ) in NM-WT preOC and cytoplasmic aggregates (yellow arrow) in NM-KA preOC. ( B ) Representative IF images for NEMO (red), LC3 (green) and NEMO-LC3 colocalization (yellow). Arrows indicate accumulation of NEMO in LC3 positive vacuole-like structures. ( C ) Representative western blot showing expression of LC3 from BMMs starved and stimulated with RANKL as shown. ( D ) Representative Western blot for mTOR expression in BMMs from NM-WT and NM-KA mice treated as shown. ( E ) Pre-osteoclasts were treated with chloroquine as indicated and number of TRAP+ multi nucleated osteoclasts (MNC) per well were counted in triplicate wells from three independent experiments (*p<0.05).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Immunofluorescence, Membrane, Western Blot, Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: BMMs from NM-WT and NM-KA mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing ISG15 and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days. ( A ) Representative TRAP staining for osteoclast (n = 6) and ( B ) quantification of TRAP positive OCs. ( C ) BMMs from NM-WT and NM-KA mice were transduced with ISG15 and pMRX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells. The cells were cultured for 2 days (preOC) followed by 6 hr of serum starvation and flow analysis to detect GFP signal or LC3 flux. ( C ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy. Blue histogram: serum starvation, yellow histogram: serum starvation + ISG15 expression ( D ) Change in Mean Fluorescent Intensity (MFI) showing LC3-GFP signal. ( E ) Wild type BMMs transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMO+/-ISG15, NEMO-K270A+/-ISG15 NEMO-WT::ISG15 (fused) and NEMO-K270A::ISG15 (fused) protein and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days.( E ) Representative TRAP staining for osteoclast (n = 3) and ( F ) quantification of TRAP positive OCs. ( G ) NEMO puncta regulation by ISG15: Live images of preOC expressing RFP-NEMO WT +/-ISG15, RFP-NEMO K270A +/-ISG15, GFP-NEMO WT ::ISG15 and GFP-NEMO K270A ::ISG15 fusion protein. Yellow arrows indicate NEMO K270A puncta. ISG15 panel which is not tagged serves as background control. ( H ) Quantification of LC3 puncta+ preOC cells shown in . ( I ) WB for LC3 in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. (*p<0.05, **p<0.01 and ***p<0.001). (::) denotes fusion. ( J ) Representative IF images (NEMO (Red); LAMP1(green)). NEMO localization in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. Green arrow- Lysosome and Yellow arrow-localization of NEMO in Lysosome. Figure 7—source data 1. quantification of TRAP positive OCs. Figure 7—source data 2. LC3-GFP FACS analysis. Figure 7—source data 3. Quantification of TRAP positive OCs. Figure 7—source data 4. Quantification of LC3 positive puncta in pre-OC cells shown in . Figure 7—source data 5. Western blot for LC3 expression in preOC expressing different NEMO and ISG15 constructs.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transduction, Generated, Retroviral, Expressing, Cell Culture, Staining, Control, Western Blot, Construct

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Representative IF images for LC3 puncta+ cells (arrow) in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein (quantified in ).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Luciferase, Recombinant, Retroviral, Plasmid Preparation, FLAG-tag, Mutagenesis, Construct, Transfection, Activity Assay, BIA-KA, Quantitation Assay, Lysis, Western Blot, Electron Microscopy, Multiplex Assay, Marker, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Sequencing, Software

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Impaired autophagy and decreased RAB7A expression are found in periodontitis patients. (A) Western blot analysis of autophagy regulator RAB7A and autophagy markers (LC3, SQSTM1) in healthy and periodontitis gingiva ( n = 3 for each group). Anti-RAB7A (cell signaling technology 95,746), anti-LC3 (cell signaling technology 12,741), anti-SQSTM1 (cell signaling technology 88,588). (B) immunofluorescence analysis of autophagy regulator RAB7A, LC3, SQSTM1, LAMP1 in lamina propria of healthy and periodontitis gingiva ( n = 3 for each group). Scale bars: 50 μm; zoom: 20 μm. Anti-RAB7A (cell signaling technology 95,746). anti-LC3 (cell signaling Technology,12741), anti-SQSTM1 (cell signaling Technology,88588). Data were presented as mean ± SEM. p -values were calculated through two-tailed Student’s t -tests.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Expressing, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: RAB7A agonist ML098 promotes autophagy and inhibits alveolar bone loss in periodontitis mice. (A) mouse experimental periodontitis scheme. The 6-8-week-old mice were randomly divided into three groups. The control group was not treated. The periodontitis group was subjected to ligature and P. g . on the maxillary second molars with or without RAB7A agonist ML098 (1 mg/kg) intraperitoneal injection (every 2 days). Mice were harvested 10 days later for periodontal tissue analysis. (B) Representative micro-ct analysis of alveolar bone in normal control mice, periodontitis mice, and periodontitis mice with ML098 group. Scale bars: 500 μm. (C) micro-ct analysis of the CEJ-ABC distance at the palatal side of the second molar (indicated by red lines) in three groups of mice ( n = 5 for each group). CEJ-ABC, the cementoenamel junction to the alveolar bone crest. (D) Representative Immunofluorescence staining of autophagy regulator RAB7A and autophagy maker LC3 and SQSTM1 in three groups of mice. Scale bars: 50 μm; zoom: 10 μm. Anti-RAB7A (cell signaling Technology,95746), anti-LC3 (cell signaling Technology,12741), anti-SQSTM1 (CST 88,588). (E, F, G) expression of RAB7A, LC3, and SQSTM1 in the gingiva of three groups of mice ( n = 5 for each group). Data were presented as mean ± SEM. p -values were calculated through one-way ANOVA test.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Control, Injection, Micro-CT, Immunofluorescence, Staining, Expressing

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Autophagy impairment is mediated by blocking autophagosome-lysosome fusion in macrophages. (A-B) time-dependent (left panel) and dose-dependent (right panel) assay of P. g .stimulation were performed on THP-1-derived macrophages. Western blot analysis was performed for the expression RAB7A and active RAB7A, autophagy markers (SQSTM1 and LC3), autophagy initiation-related proteins (BECN1, MTOR, and p-mtor). One representative blot is shown in two independent experiments. (C) GFP-RFP-LC3 THP-1 cells were stimulated with P. g . (MOI = 100), and representative time-lapse confocal images were captured to detect autophagic flux. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. Zoom, 5 μm. (D) GFP-RFP-LC3 THP-1 cells were treated with P. g . (MOI = 100), Rapa (10 nM), and Baf A1 (50 nM) for 8 h to detect autophagy flux. Representative images of fluorescent LC3 puncta were shown. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images. Data were presented as mean ± SEM. p-values were calculated through one-way ANOVA test. Rapa, rapamycin. Baf A1, bafilomycin A 1 .

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Blocking Assay, Derivative Assay, Western Blot, Expressing

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Autophagosome-lysosome fusion is blocked by decreased GTP-RAB7A. (A) Western blot analysis of total RAB7A and GTP-RAB7A (active form, immunoprecipitated by anti-GTP-RAB7A antibody) in control vs. P. g -stimulated THP-1 cells (MOI = 100, 8 h). One representative blot is shown in three independent experiments. Anti-RAB7A (cell signaling Technology,95746), anti-GTP-RAB7A (NewEast 26,923). (B) quantity analysis of the ratio of GTP-RAB7A expression to total RAB7A. (C) Western blot analysis of co-ip from THP-1 cells transfected and treated as indicated. RAB7A pulled down by GST-RILP represents GTP-RAB7A. (D) confocal microscopy analysis for colocalizations of RAB7A with RILP in control vs. P. g -stimulated THP-1 cells (MOI = 100, 8 h). RAB7A (red) and RILP (green) were stained using anti-RAB7A and anti-rilp antibodies. Scale bars: 10 μm. (E) colocalization plots report Mander’s overlap quantified from multicell images (black dots). (F) Western blot analysis of SQSTM1, LC3, total RAB7A and GTP-RAB7A (immunoprecipitated by anti-GTP-RAB7A antibody) in four groups as indicated. CID-1067700 (100 μM), ML098 (100 nM), baf A1 (50 nM). (G) GFP-RFP-LC3 THP-1 cells were treated with P. g . (MOI = 100), ML098 (100 nm), and CID-1067700 (100 μM) for 8 h and the colocalization of GFP-LC3 and RFP-LC3 was captured to detect the autophagy. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (H-I) the number of GFP + RFP + LC3 (autophagosome) and to GFP − RFP + LC3 (autolysosome) was calculated from multicell images. One representative blot is shown in two independent experiments. Data were presented as mean ± SEM. p-values were calculated through two-tailed Student’s t-tests (B, E) and one-way ANOVA test (H, I).

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Western Blot, Immunoprecipitation, Control, Expressing, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Staining, Two Tailed Test

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: USP4 promotes autophagosome-lysosome fusion by deubiquitination of RAB7A. (A) the whole lysates were extracted from THP-1 cells with or without P. g . stimulation. Lysosomal interacting proteins were immunoprecipitated and were subject to mass spectrometry. The data revealed multiple USP4 peptides in lysosome-bound protein samples, indicating the interaction between RAB7A and USP4. (B, C) Western blot analysis of Co-ip from THP-1 cells transfected and treated as indicated. (B) THP-1 cells were transfected with siRNA to knock down the USP gene. Ubiquitin-conjugated proteins were pulled down using anti-ub antibody, followed by Western blot analysis using anti-RAB7A antibody. (C) THP-1 cells were transfected with USP4 overexpression lentivirus and used GTP-RAB7A and USP4 antibodies to pull down GTP-RAB7A-binding and USP4-binding proteins to measure RAB7A (GTP-RAB7A and USP4 bounding RAB7A. (D) Representative confocal images of RAB7A with lysosome marker LAMP1. Scale bars: 10 μm. (E) LAMP1 fluorescent intensity distribution is expressed as distance along a straight line from the center of the nucleus to the peripheral area. Fluorescence intensities were quantified along a straight line extending from the center of the cell’s nucleus (designated as fractional distance = 0) to the plasma membrane (designated as fractional distance = 1.0). (F) GFP-RFP-LC3 THP-1 cells were transfected with USP4 siRNA and USP4 overexpressed lentivirus to detect autophagy flux. Representative images were captured to display the colocalization of GFP-LC3 and RFP-LC3 after being treated with P. g . (MOI = 100, 8 h). Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (G-H) the number of GFP + RFP + LC3 (autophagosome) and to GFP − RFP + LC3 (autolysosome) was calculated from multicell images. Data were presented as mean ± SEM. p-values were calculated through one-way ANOVA test.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay, Transfection, Knockdown, Ubiquitin Proteomics, Over Expression, Binding Assay, Marker, Fluorescence, Clinical Proteomics, Membrane

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Model of RAB7A ubiquitination-mediated impaired autophagosome-lysosome fusion in periodontitis pathogenesis. Research Paperhe periodontitis condition (right panel): in the absence of USP4, lack of RAB7A deubiquitination (GDP-RAB7A form) results in impaired autophagosome-lysosome fusion. The impaired autophagy leads to increased IL1B maturation and release, which contributes to periodontitis pathogenesis.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Ubiquitin Proteomics