Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A) Mice were sham injected or intracranially infected with LCMV (500 PFU) for 2 or 4 days. Brains were harvested and sectioned for immunostaining. LDs were stained with Bodipy (493/503) (green) and nuclei with DAPI (blue). (B) LD numbers were analysed via ImageJ analysis software. Error bars represent ± SEM, n = 6 replicate mice per condition. (C) To confirm the purity of isolated LDs from mice brains western blot analysis of LD lysates was performed probing for organelle markers for the endoplasmic reticulum (Calnexin), mitochondria (MFN1), peroxisomes (ACOX1) and LDs (ADRP). Results indicate enrichment of LDs and no contamination of other cellular organelles. (D) Comparative analysis of protein ID and abundance were analysed by LC-MS/MS highlighting the number of proteins significantly regulated from both whole brain lysate and isolated LD lysate derived from mice brains following 4 dpi of LCMV. Red and blue tiles indicate up- and down regulated proteins, respectively (E) Significantly upregulated proteins following LCMV infection were compared with one another to highlight distinct proteomic profiles between LDs and whole brain lysates. Red circle reflects proteins exclusive to the whole brain lysate, blue circle reflects proteins exclusive to LDs 2dpi and the yellow circle reflects proteins exclusive to LDs 4dpi. (F) Hierarchical clustering of the 18 significantly regulated LD resident proteins 4 dpi of LCMV across replicates and their respective z-scores from the groups: control 2 dpi, control 4 dpi, LCMV 2dpi and LCMV 4dpi. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. (G) Tree-plot displaying similarity clusters of the top 15 significantly enriched annotations from the gene ontology categories: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). Only annotations that surpassed an adjusted p-value of less than 0.05 were included. ( H ) Analysis of average number of LDs localised to each brain cell type with and without LCMV 4 dpi. Error bars represent values ± SEM. P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n= 64 tissue sections over 6 mice). (I) Hierarchical clustering displaying the z-scores and gene ontology between replicates of the 92 significantly regulated astrocyte derived LD proteins following dsRNA stimulation at 24 hrs. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. Gene ontology was performed using the Uniprot database within Perseus. Proteins were grouped into 5 main categories: cellular metabolism, immune response, lipid metabolism, protein transport and other. (J) Interaction diagram of significantly changed proteins following dsRNA viral mimic stimulation created using STRING confidence scores and interactions in cytoscape. Nodes are coloured in a continuous scale based off their log2 fold change, with red coloured nodes having the highest fold change and blue nodes having the greatest negative fold change. Proteins with no interactions with any other proteins were not represented within the network. All clusters are clustered using the ClusterONE plugin within cytoscape with a p < 0.05 significance threshold. (K) Similarities and differences between significantly enriched proteins from astrocyte derived LDs infected with either dsRNA or ZIKV at 24 hrs respectively. (L) Immunoblot analysis on whole cell lysate and isolated LD lysate from both mock and dsRNA stimulated astrocytes at 24 hrs confirmed the localisation of MX1, RIG-I, STAT1, STAT2, ZC3HAV1 and TRIM25 proteins on isolated LDs derived from cells that have been stimulated with dsRNA for 24 hrs. ADRP was used to confirm enrichment of purified LDs.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Injection, Infection, Immunostaining, Staining, Software, Isolation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Purification

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A ) Schematic of the interferon signalling pathway following viral infection with proteins absent from the LD proteome in white, proteins present on the LD proteome in grey and proteins upregulated on the LD in red. (B) Astrocyte cells were transfected with a mCherry tagged viperin, STAT1 and STAT2 (red) and cells were stained with Bodipy (493/503) to visualise LDs (green) and DAPI to visualise cell nuclei (blue). The zoomed images indicate interaction between the respective mCherry tagged proteins and LDs. Scale bar, 50μm. (C) Confocal microscopy of astrocytes transfected with mCherry, viperin-mCherry, STAT1-mCherry and STAT2-mCherry respectively, with LDs then isolated and purified. Purified LDs were then imaged to determine the interaction between viperin, STAT1 and STAT2 with LDs. The zoom panel highlights the localisation of the aforementioned proteins with isolated and purified LDs. Scale bar, 400 μm for transfection panel, 50 μm for merge and 20 μm for zoom panels. (D) Isolated LDs were also imaged to allow the quantification of the percentage of LDs that have mCherry co-localisation with each data point representing the average percentage of co-localisation in a field of view of ∼500-3000 LDs (n=18-25) over 2 independent experiments. Error bars represent ± SEM. (E) To further validate the confocal microscopy of isolated LDs the purified LDs and the whole cell protein from the transfected astrocytes were immunoblotted for mCherry to show detection of mCherry tagged proteins (F) LD-STAT1 co-localisation events were visualised post dsRNA transfection via super resolution microscopy: Single-molecule localization microscopy (SMLM). The epifluorescence images of LDs were merged with the SMLM images of STAT1 (STAT1, phSTAT1 (S; serine)/ (T; tyrosine), and co-localisation events counted across a 72 hrs dsRNA stimulation time course. Cells were immunolabelled and imaged for LDs (Bodipy (493/503), epi, blue) and STAT1 (AlexaFluor647, SMLM, yellow). (n = 3 independent biological replicates with >150 cells imaged). Scale bars represent 1µm, with zoom inserts representing 1µm x 1µm in size. (G) LD-STAT1 co-localisation data was quantified as number of co-localisation events per cell. Captured images were merged and analysed using the ThunderSTORM plugin in ImageJ for co-localisation events. Error bars represent ± SEM, n= 6 cells over 3 independent assays.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Infection, Transfection, Staining, Confocal Microscopy, Isolation, Purification, Super-Resolution Microscopy, Microscopy

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A) Mice were sham injected or intracranially infected with LCMV (500 PFU) for 2 or 4 days. Brains were harvested and sectioned for immunostaining. LDs were stained with Bodipy (493/503) (green) and nuclei with DAPI (blue). (B) LD numbers were analysed via ImageJ analysis software. Error bars represent ± SEM, n = 6 replicate mice per condition. (C) To confirm the purity of isolated LDs from mice brains western blot analysis of LD lysates was performed probing for organelle markers for the endoplasmic reticulum (Calnexin), mitochondria (MFN1), peroxisomes (ACOX1) and LDs (ADRP). Results indicate enrichment of LDs and no contamination of other cellular organelles. (D) Comparative analysis of protein ID and abundance were analysed by LC-MS/MS highlighting the number of proteins significantly regulated from both whole brain lysate and isolated LD lysate derived from mice brains following 4 dpi of LCMV. Red and blue tiles indicate up- and down regulated proteins, respectively (E) Significantly upregulated proteins following LCMV infection were compared with one another to highlight distinct proteomic profiles between LDs and whole brain lysates. Red circle reflects proteins exclusive to the whole brain lysate, blue circle reflects proteins exclusive to LDs 2dpi and the yellow circle reflects proteins exclusive to LDs 4dpi. (F) Hierarchical clustering of the 18 significantly regulated LD resident proteins 4 dpi of LCMV across replicates and their respective z-scores from the groups: control 2 dpi, control 4 dpi, LCMV 2dpi and LCMV 4dpi. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. (G) Tree-plot displaying similarity clusters of the top 15 significantly enriched annotations from the gene ontology categories: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). Only annotations that surpassed an adjusted p-value of less than 0.05 were included. ( H ) Analysis of average number of LDs localised to each brain cell type with and without LCMV 4 dpi. Error bars represent values ± SEM. P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n= 64 tissue sections over 6 mice). (I) Hierarchical clustering displaying the z-scores and gene ontology between replicates of the 92 significantly regulated astrocyte derived LD proteins following dsRNA stimulation at 24 hrs. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. Gene ontology was performed using the Uniprot database within Perseus. Proteins were grouped into 5 main categories: cellular metabolism, immune response, lipid metabolism, protein transport and other. (J) Interaction diagram of significantly changed proteins following dsRNA viral mimic stimulation created using STRING confidence scores and interactions in cytoscape. Nodes are coloured in a continuous scale based off their log2 fold change, with red coloured nodes having the highest fold change and blue nodes having the greatest negative fold change. Proteins with no interactions with any other proteins were not represented within the network. All clusters are clustered using the ClusterONE plugin within cytoscape with a p < 0.05 significance threshold. (K) Similarities and differences between significantly enriched proteins from astrocyte derived LDs infected with either dsRNA or ZIKV at 24 hrs respectively. (L) Immunoblot analysis on whole cell lysate and isolated LD lysate from both mock and dsRNA stimulated astrocytes at 24 hrs confirmed the localisation of MX1, RIG-I, STAT1, STAT2, ZC3HAV1 and TRIM25 proteins on isolated LDs derived from cells that have been stimulated with dsRNA for 24 hrs. ADRP was used to confirm enrichment of purified LDs.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Injection, Infection, Immunostaining, Staining, Software, Isolation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Purification

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A ) Schematic of the interferon signalling pathway following viral infection with proteins absent from the LD proteome in white, proteins present on the LD proteome in grey and proteins upregulated on the LD in red. (B) Astrocyte cells were transfected with a mCherry tagged viperin, STAT1 and STAT2 (red) and cells were stained with Bodipy (493/503) to visualise LDs (green) and DAPI to visualise cell nuclei (blue). The zoomed images indicate interaction between the respective mCherry tagged proteins and LDs. Scale bar, 50μm. (C) Confocal microscopy of astrocytes transfected with mCherry, viperin-mCherry, STAT1-mCherry and STAT2-mCherry respectively, with LDs then isolated and purified. Purified LDs were then imaged to determine the interaction between viperin, STAT1 and STAT2 with LDs. The zoom panel highlights the localisation of the aforementioned proteins with isolated and purified LDs. Scale bar, 400 μm for transfection panel, 50 μm for merge and 20 μm for zoom panels. (D) Isolated LDs were also imaged to allow the quantification of the percentage of LDs that have mCherry co-localisation with each data point representing the average percentage of co-localisation in a field of view of ∼500-3000 LDs (n=18-25) over 2 independent experiments. Error bars represent ± SEM. (E) To further validate the confocal microscopy of isolated LDs the purified LDs and the whole cell protein from the transfected astrocytes were immunoblotted for mCherry to show detection of mCherry tagged proteins (F) LD-STAT1 co-localisation events were visualised post dsRNA transfection via super resolution microscopy: Single-molecule localization microscopy (SMLM). The epifluorescence images of LDs were merged with the SMLM images of STAT1 (STAT1, phSTAT1 (S; serine)/ (T; tyrosine), and co-localisation events counted across a 72 hrs dsRNA stimulation time course. Cells were immunolabelled and imaged for LDs (Bodipy (493/503), epi, blue) and STAT1 (AlexaFluor647, SMLM, yellow). (n = 3 independent biological replicates with >150 cells imaged). Scale bars represent 1µm, with zoom inserts representing 1µm x 1µm in size. (G) LD-STAT1 co-localisation data was quantified as number of co-localisation events per cell. Captured images were merged and analysed using the ThunderSTORM plugin in ImageJ for co-localisation events. Error bars represent ± SEM, n= 6 cells over 3 independent assays.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Infection, Transfection, Staining, Confocal Microscopy, Isolation, Purification, Super-Resolution Microscopy, Microscopy

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A) Mice were sham injected or intracranially infected with LCMV (500 PFU) for 2 or 4 days. Brains were harvested and sectioned for immunostaining. LDs were stained with Bodipy (493/503) (green) and nuclei with DAPI (blue). (B) LD numbers were analysed via ImageJ analysis software. Error bars represent ± SEM, n = 6 replicate mice per condition. (C) To confirm the purity of isolated LDs from mice brains western blot analysis of LD lysates was performed probing for organelle markers for the endoplasmic reticulum (Calnexin), mitochondria (MFN1), peroxisomes (ACOX1) and LDs (ADRP). Results indicate enrichment of LDs and no contamination of other cellular organelles. (D) Comparative analysis of protein ID and abundance were analysed by LC-MS/MS highlighting the number of proteins significantly regulated from both whole brain lysate and isolated LD lysate derived from mice brains following 4 dpi of LCMV. Red and blue tiles indicate up- and down regulated proteins, respectively (E) Significantly upregulated proteins following LCMV infection were compared with one another to highlight distinct proteomic profiles between LDs and whole brain lysates. Red circle reflects proteins exclusive to the whole brain lysate, blue circle reflects proteins exclusive to LDs 2dpi and the yellow circle reflects proteins exclusive to LDs 4dpi. (F) Hierarchical clustering of the 18 significantly regulated LD resident proteins 4 dpi of LCMV across replicates and their respective z-scores from the groups: control 2 dpi, control 4 dpi, LCMV 2dpi and LCMV 4dpi. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. (G) Tree-plot displaying similarity clusters of the top 15 significantly enriched annotations from the gene ontology categories: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). Only annotations that surpassed an adjusted p-value of less than 0.05 were included. ( H ) Analysis of average number of LDs localised to each brain cell type with and without LCMV 4 dpi. Error bars represent values ± SEM. P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n= 64 tissue sections over 6 mice). (I) Hierarchical clustering displaying the z-scores and gene ontology between replicates of the 92 significantly regulated astrocyte derived LD proteins following dsRNA stimulation at 24 hrs. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. Gene ontology was performed using the Uniprot database within Perseus. Proteins were grouped into 5 main categories: cellular metabolism, immune response, lipid metabolism, protein transport and other. (J) Interaction diagram of significantly changed proteins following dsRNA viral mimic stimulation created using STRING confidence scores and interactions in cytoscape. Nodes are coloured in a continuous scale based off their log2 fold change, with red coloured nodes having the highest fold change and blue nodes having the greatest negative fold change. Proteins with no interactions with any other proteins were not represented within the network. All clusters are clustered using the ClusterONE plugin within cytoscape with a p < 0.05 significance threshold. (K) Similarities and differences between significantly enriched proteins from astrocyte derived LDs infected with either dsRNA or ZIKV at 24 hrs respectively. (L) Immunoblot analysis on whole cell lysate and isolated LD lysate from both mock and dsRNA stimulated astrocytes at 24 hrs confirmed the localisation of MX1, RIG-I, STAT1, STAT2, ZC3HAV1 and TRIM25 proteins on isolated LDs derived from cells that have been stimulated with dsRNA for 24 hrs. ADRP was used to confirm enrichment of purified LDs.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Injection, Infection, Immunostaining, Staining, Software, Isolation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Purification

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A) Mice were sham injected or intracranially infected with LCMV (500 PFU) for 2 or 4 days. Brains were harvested and sectioned for immunostaining. LDs were stained with Bodipy (493/503) (green) and nuclei with DAPI (blue). (B) LD numbers were analysed via ImageJ analysis software. Error bars represent ± SEM, n = 6 replicate mice per condition. (C) To confirm the purity of isolated LDs from mice brains western blot analysis of LD lysates was performed probing for organelle markers for the endoplasmic reticulum (Calnexin), mitochondria (MFN1), peroxisomes (ACOX1) and LDs (ADRP). Results indicate enrichment of LDs and no contamination of other cellular organelles. (D) Comparative analysis of protein ID and abundance were analysed by LC-MS/MS highlighting the number of proteins significantly regulated from both whole brain lysate and isolated LD lysate derived from mice brains following 4 dpi of LCMV. Red and blue tiles indicate up- and down regulated proteins, respectively (E) Significantly upregulated proteins following LCMV infection were compared with one another to highlight distinct proteomic profiles between LDs and whole brain lysates. Red circle reflects proteins exclusive to the whole brain lysate, blue circle reflects proteins exclusive to LDs 2dpi and the yellow circle reflects proteins exclusive to LDs 4dpi. (F) Hierarchical clustering of the 18 significantly regulated LD resident proteins 4 dpi of LCMV across replicates and their respective z-scores from the groups: control 2 dpi, control 4 dpi, LCMV 2dpi and LCMV 4dpi. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. (G) Tree-plot displaying similarity clusters of the top 15 significantly enriched annotations from the gene ontology categories: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). Only annotations that surpassed an adjusted p-value of less than 0.05 were included. ( H ) Analysis of average number of LDs localised to each brain cell type with and without LCMV 4 dpi. Error bars represent values ± SEM. P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n= 64 tissue sections over 6 mice). (I) Hierarchical clustering displaying the z-scores and gene ontology between replicates of the 92 significantly regulated astrocyte derived LD proteins following dsRNA stimulation at 24 hrs. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. Gene ontology was performed using the Uniprot database within Perseus. Proteins were grouped into 5 main categories: cellular metabolism, immune response, lipid metabolism, protein transport and other. (J) Interaction diagram of significantly changed proteins following dsRNA viral mimic stimulation created using STRING confidence scores and interactions in cytoscape. Nodes are coloured in a continuous scale based off their log2 fold change, with red coloured nodes having the highest fold change and blue nodes having the greatest negative fold change. Proteins with no interactions with any other proteins were not represented within the network. All clusters are clustered using the ClusterONE plugin within cytoscape with a p < 0.05 significance threshold. (K) Similarities and differences between significantly enriched proteins from astrocyte derived LDs infected with either dsRNA or ZIKV at 24 hrs respectively. (L) Immunoblot analysis on whole cell lysate and isolated LD lysate from both mock and dsRNA stimulated astrocytes at 24 hrs confirmed the localisation of MX1, RIG-I, STAT1, STAT2, ZC3HAV1 and TRIM25 proteins on isolated LDs derived from cells that have been stimulated with dsRNA for 24 hrs. ADRP was used to confirm enrichment of purified LDs.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Injection, Infection, Immunostaining, Staining, Software, Isolation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Purification

Journal: bioRxiv

Article Title: Virally induced lipid droplets are a platform for innate immune signalling complexes

doi: 10.1101/2023.04.20.537741

Figure Lengend Snippet: (A) Mice were sham injected or intracranially infected with LCMV (500 PFU) for 2 or 4 days. Brains were harvested and sectioned for immunostaining. LDs were stained with Bodipy (493/503) (green) and nuclei with DAPI (blue). (B) LD numbers were analysed via ImageJ analysis software. Error bars represent ± SEM, n = 6 replicate mice per condition. (C) To confirm the purity of isolated LDs from mice brains western blot analysis of LD lysates was performed probing for organelle markers for the endoplasmic reticulum (Calnexin), mitochondria (MFN1), peroxisomes (ACOX1) and LDs (ADRP). Results indicate enrichment of LDs and no contamination of other cellular organelles. (D) Comparative analysis of protein ID and abundance were analysed by LC-MS/MS highlighting the number of proteins significantly regulated from both whole brain lysate and isolated LD lysate derived from mice brains following 4 dpi of LCMV. Red and blue tiles indicate up- and down regulated proteins, respectively (E) Significantly upregulated proteins following LCMV infection were compared with one another to highlight distinct proteomic profiles between LDs and whole brain lysates. Red circle reflects proteins exclusive to the whole brain lysate, blue circle reflects proteins exclusive to LDs 2dpi and the yellow circle reflects proteins exclusive to LDs 4dpi. (F) Hierarchical clustering of the 18 significantly regulated LD resident proteins 4 dpi of LCMV across replicates and their respective z-scores from the groups: control 2 dpi, control 4 dpi, LCMV 2dpi and LCMV 4dpi. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. (G) Tree-plot displaying similarity clusters of the top 15 significantly enriched annotations from the gene ontology categories: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). Only annotations that surpassed an adjusted p-value of less than 0.05 were included. ( H ) Analysis of average number of LDs localised to each brain cell type with and without LCMV 4 dpi. Error bars represent values ± SEM. P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n= 64 tissue sections over 6 mice). (I) Hierarchical clustering displaying the z-scores and gene ontology between replicates of the 92 significantly regulated astrocyte derived LD proteins following dsRNA stimulation at 24 hrs. Blue tiles refer to proteins with a z-score < 0 with red tiles referring to proteins with a z-score > 0. Gene ontology was performed using the Uniprot database within Perseus. Proteins were grouped into 5 main categories: cellular metabolism, immune response, lipid metabolism, protein transport and other. (J) Interaction diagram of significantly changed proteins following dsRNA viral mimic stimulation created using STRING confidence scores and interactions in cytoscape. Nodes are coloured in a continuous scale based off their log2 fold change, with red coloured nodes having the highest fold change and blue nodes having the greatest negative fold change. Proteins with no interactions with any other proteins were not represented within the network. All clusters are clustered using the ClusterONE plugin within cytoscape with a p < 0.05 significance threshold. (K) Similarities and differences between significantly enriched proteins from astrocyte derived LDs infected with either dsRNA or ZIKV at 24 hrs respectively. (L) Immunoblot analysis on whole cell lysate and isolated LD lysate from both mock and dsRNA stimulated astrocytes at 24 hrs confirmed the localisation of MX1, RIG-I, STAT1, STAT2, ZC3HAV1 and TRIM25 proteins on isolated LDs derived from cells that have been stimulated with dsRNA for 24 hrs. ADRP was used to confirm enrichment of purified LDs.

Article Snippet: The primary antibodies used were: mouse monoclonal αCalnexin (1:1000; sc-23954, Santa Cruz Biotechnology), mouse monoclonal αmfn1 (1:1000; sc-166644, Santa Cruz Biotechnology), mouse monoclonal αACOX1 (1:1000; sc-517306, Santa Cruz Biotechnology), rabbit monoclonal αPerilipin-2 (1:2000; ab108323, Abcam), mouse monoclonal αRIG-I (1:1000; sc-376845 Santa Cruz Biotechnology), rabbit monoclonal αMX1 (1:2000; ab207414, Abcam), rabbit polyclonal αSTAT1 (1:1000; 9172, Cell Signaling Technology), rabbit monoclonal αSTAT2 (1:1000; A3588, ABclonal), rabbit polyclonal αZC3HAV1 (1:5000; ab154680, Abcam), rabbit monoclonal αPhospho-STAT1 (Tyr701) (1:1000; #7649, Cell Signaling Technology), rabbit polyclonal αmCherry (1:1000; 5993, BioVision) and rabbit polyclonal αTRIM25 (1:1000; ab86365, Abcam).

Techniques: Injection, Infection, Immunostaining, Staining, Software, Isolation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Purification