sim-a9 wild type  (Absolute Biotech Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: Video 7 Motor function recovery of mice in AAV-FBXL12 delivery group

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: Video 9 BMS evaluation of AAV-Fbxl12 group mice

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: m6A methylation of Fbxl12 is a candidate master process during SCI progression. a Schematic illustration of the anatomical location of the C57BL/6 mouse spinal cord crush injury model. b Normalized peak areas and modification proportions of various mRNAs in the sham and SCI groups. The normalized peak area was calculated as the number of sites detected for this type of modification, and the modification proportion was calculated as the ratio of the detected sites for one type of modification among all detected modification sites. c Immunoblotting of m6A writers and erasers in spinal cord tissue at different days postinjury as indicated. d Schematic diagram of the analysis of integrated transcriptional and epitranscriptomic profiling. e Gene cluster dendrogram of the WGCNA results. f Volcano plot of gene expression at 7 days postinjury compared with that in the sham group. g mRNA expression and m6A level of Fbxl12 at different days postinjury as indicated (one-way ANOVA, * P < 0.05, ** P < 0.01). h Correlation between Fbxl12 mRNA expression and Fbxl12 m6A levels. i RT‒qPCR of Fbxl12 mRNA at different days postinjury as indicated ( t test, mean ± SEM; all groups compared with the Sham group, * P < 0.05, ** P < 0.01, *** P < 0.001). j Immunoblotting of FBXL12 in spinal cord tissue at different days postinjury, as indicated. The results are representative of three independent experiments

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Methylation, Modification, Western Blot, Gene Expression, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: m6A promotes FBXL12 synthesis in microglia. a Images of spinal cord lesions at different time points after injury; the lesions were stained with antibodies against FBXL12 (green) and IBA1 (red) or with DAPI (blue). b Quantification of IBA1- and IBA1/FBXL12-positive cells within the 500 µm range at different time points after injury. (one-way ANOVA, mean ± SEM; *, IBA1 positive, ** P < 0.01, **** P < 0.0001, n = 5; #, IBA1/FBXL12 positive, # P < 0.05, #### P < 0.0001, n = 5; all groups compared with the Sham group). c , d Immunoblotting of wild-type microglia treated with MBP and LPS as indicated. The graph below shows the blots normalized to β-actin (mean ± SEM, n = 3). e Immunoblotting of wild-type microglia treated with 200 ng/mL MBP for the indicated times. The graph below shows the blots normalized to β-actin (one-way ANOVA, mean ± SEM; *** P < 0.001, **** P < 0.0001, n = 3). f RT‒qPCR quantitation of Fbxl12 mRNA expression in SIM-A9 cells treated with 200 ng/mL MBP for the indicated times. mRNA expression is relative to that of the control group (mean ± SEM, n = 3). g MeRIP‒qPCR quantitation of Fbxl12 mRNA m6A levels in SIM-A9 cells treated with 200 ng/mL MBP for the indicated times. The m6A modification level is relative to that of the control group ( t test, mean ± SEM; ** P < 0.01, n = 3). h Immunoblotting of FBXL2 expression in microglia with METTL3, METTL14 and YTHDF1 knockdown as indicated. i Images of spinal sections at 28 dpi from different groups stained with antibodies against IBA1 and Mettl3 or with DAPI. j Images of spinal sections at 28 dpi from different groups stained with antibodies against IBA1 and Fbxl12 or with DAPI. k Quantification of IBA1 (left) and IBA1/Mettl3 (right) positive cells at 28 dpi (one-way ANOVA, mean ± SEM; **** P < 0.0001, n = 4–5). l Quantification of Fbxl12 immunoreactive intensity around the lesion site (left) and microglia (right) at 28 dpi ( t test, mean ± SEM; *** P < 0.001, n = 6–8)

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Staining, Western Blot, Quantitation Assay, Expressing, Control, Modification, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: FBXL12 regulates cytoskeletal reorganization, promotes migration and regulates the immune response of microglia. a Images of different microglia stained with crystal violet. Where indicated, the cells were wild-type (WT), Fbxl12-overexpressing (Fbxl12 WT-OE ), Fbxl12-mutant (Fbxl12 Mut-OE ) and Fbxl2-deficient (Fbxl12 -/- ). b Quantification of migrated microglia in ( a ) (one-way ANOVA, *** P < 0.001, n = 3). c SEM images of different microglia. d Images of different microglia stained with F-actin and DAPI. Representative microglia are shown below the magnified window. Scale bar as indicated. e Quantification of filopodium numbers (top, n = 5) and lengths (bottom, n = 30) of microglia in ( d ) (one-way ANOVA, * P < 0.05, *** P < 0.001). f Chemokine secretion in microglia (Fbxl12 WT-OE vs WT). ( t test, mean ± SEM; * P < 0.05, *** P < 0.001, n = 3). g Volcano plot of differentially expressed genes in microglia (Fbxl12 WT-OE vs WT). h Selected KEGG pathways differentially enriched in WT versus Fbxl12 WT-OE microglia. i Heatmap of differentially expressed genes associated with positive regulation of the migration pathway in microglia (Fbxl12 WT-OE vs WT). j Gene Ontology (GO) terms enriched in microglia (Fbxl12 WT-OE vs WT) were selected. k , l Heatmap of differentially expressed genes associated with inflammation and chemokine terms and complement and coagulation cascades ( l ) in microglia (Fbxl12 WT-OE vs WT)

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Migration, Staining, Mutagenesis, Coagulation

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: AAV-Fbxl12 delivery promotes microglial migration after SCI. a Schematic description of the intrathecal delivery of AAV-Fbxl12. b Immunofluorescence staining of spinal cord tissue for FBXL12 and IBA1 at 28 dpi. c Images of spinal cord lesions stained with antibodies against IBA1 and Fbxl12 or with DAPI (blue) and quantification of Fbxl12 immunoreactive intensity in microglia at 28 dpi ( t test, mean ± SEM; *** P < 0.001, n = 3–6). d Images of the spinal cord stained with IBA1 (4 mm in length centered around the injury site). e Quantification of IBA1 immunoreactive intensity ( n = 5). f Uniform manifold approximation and projection (UMAP) plot of spots from all sections visualized via the Seurat package. Through unsupervised clustering and marker gene expression, 7 cell types were annotated. The proportions of various cell types in the SCI and SCI Fbxl12 -OE groups are presented in pie charts (left). g–i Expression profiles of marker genes of neurons, microglia and fibroblasts are presented via UMAP, where each dot represents an individual spot and the color represents the expression level (dark red, high expression; dark blue, low). Spatial distribution of the expression profiles of marker genes in spinal cord sections, with colors representing expression levels (dark blue, high expression; dark blue, low expression). j UMAP plot of microglia with other cell types denoted by gray (top) and spatial distribution (bottom) of GSVA scores for positive regulation of migration. k Images of spinal sections at 28 dpi stained with antibodies against IBA1 and IFITM3 ( n = 5). l Quantification of IBA1- and IBA1/IFITM3-positive cells within the 1500 µm range at different time points after injury. ( t test, mean ± SEM; *, IBA1 positive, ** P < 0.01, n = 5; #, IBA1/IFITM3 positive, # P < 0.05, n = 5). m UMAP plot of microglia with other cell types denoted by gray (up) and spatial distribution (down) of GSVA scores for scar-free wound healing promotion

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Migration, Immunofluorescence, Staining, Marker, Gene Expression, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: Intrathecal delivery of FBXL12 to microglia reduces scarring, ameliorates pathology and improves axon regeneration in mice. a , b Images of spinal sections at 28 and 56 dpi stained with antibodies against the indicated proteins. Quantification of the indicated immunoreactive area in the lesion site below the images (dashed area in images) ( t test, mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 5–8). c Images of anti-5-HT-stained spinal sections from different groups of mice at 28 dpi and 56 dpi, showing serotonergic axons. Yellow stars indicate the lesion site. d Quantification of the density of serotonergic axons (normalized to the density proximal to the lesion site) in the spinal cord distal to the lesion site at 56 dpi (two-way ANOVA, mean ± SEM; * P < 0.05, **** P < 0.0001, n = 5–7). e Orthogonal projection image of the spinal cord with tissue clearing and 3D imaging. f Images of spinal sections at 28 dpi stained with antibodies against CD206, iNOS, S100A10, NF200, NESTIN and SOX2. g Amplitude and latency period of MEPs at 56 days postinjury ( t test, mean ± SEM; * P < 0.05, n = 3). h Representative images of hindlimb movement in mice at 56 dpi with or without AAV-FBXL12 overexpression. i BMSs of mice with or without AAV-FBXL12 overexpression (two-way ANOVA, mean ± SEM; * P < 0.05, ** P < 0.01, n = 10)

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Staining, Imaging, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: F-box/LRR-repeat protein 12 reorchestrated microglia to inhibit scarring and achieve adult spinal cord injury repair

doi: 10.1038/s41392-025-02354-0

Figure Lengend Snippet: FBXL12 mediates MYH14 K63 ubiquitination to orchestrate the cytoskeletal reorganization of microglia. a Schematic description of immunoprecipitation‒mass spectrometry (IP‒MS) of microglia treated with 200 ng/mL MBP for 4 days. b , c GO-molecular function and cellular component analyses of whole FBXL12-binding proteins. d KEGG pathway analysis of MBP-treated microglia. e Volcano plot of FBP changes in wild-type microglia and MBP-treated microglia. f Immunoblotting of MYH14 and MYH9 in different microglia. g Immunoblotting of MYH14 in WT and Fbxl12 −/− microglia treated with 200 ng/mL MBP for the indicated times. h Immunoprecipitation of FBXL12 with MYH14 in WT and Fbxl12 −/− microglia. i Images of different microglia stained with F-actin and antibodies against MYH14. Representative microglia are shown in the middle and below magnified windows. Scale bar as indicated. j Crystal violet staining of WT and Fbxl12 WT-OE microglia. k Quantification of migrated microglia in j (one-way ANOVA, mean ± SEM; *** P < 0.001, **** P < 0.0001, n = 3). l Images of WT and Fbxl12 WT-OE microglia stained with F-actin, with or without MYH14. m Quantification of the number (left, n = 6) and length (right, n = 16) of filopodium in l (one-way ANOVA, mean ± SEM; **** P < 0.0001)

Article Snippet: SIM-A9 Fbxl12−/− , SIM-A9 Fbxl12 wild-type-overexpressing (Fbxl12 WT-OE ), and SIM-A9 Fbxl12 mutant-overexpressing (Fbxl12 Mut-OE ) strains were obtained from Cyagen Biosciences.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A , B IL-1β release measured via ELISA from supernatants of SIM-A9 cells 24 h after manipulation of SKA2 and/or FKBP5 expression, and following overnight LPS (100 ng/mL) and treatment with LLOMe (0.25 mM) for 3 h (unpaired two tailed t-test: (A) t 4 = 11.99, p = 0.0003; one-way ANOVA: B F 3, 8 = 158.6, p < 0.0001; Tukey’s post hoc test: ctrl vs. SKA2-OE, p = 0.0384, ctrl vs. FKBP5-OE, p < 0.0001, SKA2-OE vs. FKBP5-OE, p < 0.0001, FKBP5-OE vs. SKA2 + FKBP5 OE, p < 0.0001; n = mean derived from three independent in vitro experiments). C Schematic overview of the SA pathway with SKA2 and FKBP5. The cargo receptor TRIM16, together with SEC22B, transfers molecular cargo (e.g., IL-1β) to the autophagy-related LC3B-positive membrane carriers. SEC22B, now acting as an R-SNARE on the delimiting membrane facing the cytosol, carries out fusion at the plasma membrane in conjunction with the Q bc -SNAREs, SNAP23 and SNAP29 (SNAP23/29), and one of the plasma membrane Q a -SNAREs, STX3 or STX4 (STX3/4), thus delivering IL-1β to the extracellular milieu, where it exerts its biological functions. FKBP5 acts as a positive regulator of SA by enhancing TRIM16-SEC22B complex formation as well as autophagosome-plasma membrane fusion via the SNARE-protein complex assembly. In contrast, SKA2 inhibits the SNARE-protein complex formation during vesicle-plasma membrane fusion, thereby acting as gatekeeper of SA. D , E Schematic overview of in vivo microdialysis and the experimental design and timeline; each sample was collected over 30 min indicated by the light gray lines. Quantifications of IL-1β, determined by capillary-based immunoblotting from in vivo medioprefrontal cortex microdialysis of C57Bl/6NCrl mice injected intraperitoneally with ULK1 inhibitor (ULK1i, an autophagy inhibitor) or saline ( F ; repeated measures two-way ANOVA, time × treatment interaction: F 5, 30 = 7.064, p = 0.0002; Šidák’s multiple comparisons post hoc test, post-FS-1: p = 0.0084; n = 4 mice per group) as well as of wild type (WT) and global Fkbp5 knockout mice ( G ; repeated measures two-way ANOVA, time × genotype interaction: F 5, 30 = 34.15, p < 0.0001; Šidák’s multiple comparisons post hoc test: FS: p = 0.009, post-FS-1: p = 0.0163, post-FS-2: p = 0.0294; n = 4 mice per group). FS foot shock. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Derivative Assay, In Vitro, Membrane, In Vivo, Western Blot, Injection, Saline, Knock-Out

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SIM-A9 Sec22b −/− cells expressing ASC (apoptosis-associated speck-like protein containing a CARD) -mCerulean (via epifluorescence) show a significantly decreased number of intracellular (white arrows) ASC specks compared to wild type (WT) SIM-A9 cells (unpaired two tailed t-test: t 4 = 3.206, p = 0.0327; n = mean derived from three independent in vitro experiments). B In WT SIM-A9 cells knockdown of Ska2 or LPS treatment leads to a significantly increased number of intracellular ASC specks compared to Scr-shRNA or LPS-treated cells (2-way ANOVA: main LPS treatment effect ($), F 1,31 = 10.60, p = 0.0027, main Ska2 knockdown effect (*), F 1,31 = 5.482, p = 0.0258; n = 9 WT Veh SCR-shRNA, n = 9 WT Veh SKA2-shRNA, n = 9 WT LPS SCR-shRNA, n = 8 WT LPS SKA2-shRNA). C In contrast, knockdown of Ska2 or LPS treatment does not have any effects on the number of ASC specks in SIM-A9 Sec22b −/− cells (2-way ANOVA: n. s. treatment effect F 1,29 = 0.312, p = 0.5804, main Ska2 knockdown effect, F 1,29 = 0.055, p = 0.8157; n = 9 for SEC22B KO Veh SCR-shRNA and SKA2-shRNA, n = 7 SEC22B KO LPS SCR-shRNA, n = 8 SEC22B KO LPS SKA2-shRNA). D , E Knockdown of Ska2 leads to significantly increased SEC22B binding to SNAP29 (unpaired two tailed t-test: t 4 = 4.113, p = 0.0063; n = 4 independent biological replicates) as well as NEK7 binding to NLRP3 in protein lysates of organotypic hippocampal slice cultures (unpaired two tailed t-test: t 4 = 2.998, p = 0.0241; n = 4 independent biological replicates). F IHC images of ASC (green) and DAPI (blue) 2 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus. Quantification of ASC+ cells (left) and ASC specks (right) 2 weeks after viral injection (paired t-test: ASC+ cells, t 2 = 6.414, p = 0.0235, ASC specks, t 2 = 6.937, p = 0.0202; n = 3 mice). G IHC images of ASC (green) and DAPI (blue) 4 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus. Quantification of ASC+ cells (left) and ASC specks (right) 4 weeks after viral injection (paired t-test: ASC+ cells, t 2 = 8.511, p = 0.0135; ASC specks, t 2 = 10.99, p = 0.0082; n = 3 mice). H IHC images of CASPASE-1 (CASP-1) (green) and mCherry (red, viral marker) 2 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus (left). (right) Quantification of CASP-1 expression 2 weeks after viral injection (paired t-test: t 3 = 2.842, p = 0.0655, n = 4 mice). I IHC images of CASP-1 (green) and mCherry (red, viral marker) 4 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus (left). (right) Quantification of CASP-1 expression 4 weeks after viral injection (paired t-test: t 3 = 3.367, p = 0.0435, n = 4 mice). J Full length Gasdermin D (GSDMD FL) levels as well as the ratio of the cleaved N-terminal form of GSDMD (GSDMD N-term) to GSDMD FL are increased 2 weeks after Ska2 knockdown (unpaired two tailed t-test; GSDMD FL/ β-actin: t 18 = 4.105, p = 0.0007, GSDMD N-term/GSDMD FL: t 18 = 9.259, p < 0.0001; n = 10 independent biological replicates per group). K Examples blots of ( E ). L Schematic overview of the interaction between secretory autophagy (SA) and the GSDMD-mediated IL-1β release. SKA2 depletion results in increased SA-dependent IL-1β release, serving as a molecular vicious feed-forward loop for inflammasome activation. Inflammasome assembly activates CASP-1 enzymatic function. ASC in the inflammasome complex recruits CASP-1. Activation of CASP-1 cleaves GSDMD to release the N-terminal domain, which forms pores in the plasma membrane for uncontrolled IL-1β release. * = p < 0.05; ** = p < 0.01; *** = p < 0.001, **** = p < 0.0001. Data are presented as mean + SEM. Scale bar represents 5 µm in A, 50 µm in ( F , G ) (left), 10 µm in ( B , F , G ) (right), and 250 µm in ( H , I ). Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Expressing, Two Tailed Test, Derivative Assay, In Vitro, shRNA, Binding Assay, Injection, Marker, Activation Assay, Membrane