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