Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a) Venn diagrams showing the number of DEGs from analyzed SLAM-Seq data obtained with SIM-A9 cells exposed to 5 μg/mL TDP-43 aggregates (TDP43) or sham aggregates (Sham) for 4 h, pre-treated for 1 h with 10 μM C13 (or DMSO as vehicle). Results are from three independent experiments (N=3). Indicated are the number of DEGs for ‘C13-Sham vs. Sham’ and ‘C13-TDP43 vs. TDP43’ comparisons (pAdj.<0.05). b) Predicted network analysis based on the two sets of identified DEGs by using GeneMANIA . Blue labels correspond to top DEGs and red labels, to inferred genes. c) Heatmap representing the relative expression profiles (DEGs with pAdj.<0.05) comparing ‘TDP43 vs. Sham’ and ‘C13-TDP43 vs. TDP43’ treatments. The differential regulation in gene expression changes between both comparisons indicates an effect of MOK inhibitor C13 in transcriptional profile of microglial cells upon exposure to TDP-43 aggregates. d) Schematic representation of LC-MS/MS analysis of eluates from anti-phospho-Ser/Thr immunoprecipitation assays of lysates from primary microglial cells exposed to 5 μg/mL TDP-43 aggregates, pre-treated for 1 h with 10 μM C13 (or DMSO as vehicle).

Article Snippet: MOK-knockout (MOK-KO) SIM-A9 cell clones (H14 and A7) were generated by CRISPR/Cas9 technology (Synthego, Redwood City, CA, USA).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation

Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a) Quantification of pro-inflammatory cytokines by ELISA in supernatants from primary microglial cells stimulated with 1 μg/mL LPS for 5 h (TNFα) or 24 h (IL-6, IL-1β) after pre-treatment with 10 μM C13 (or DMSO) for 1 h (N=4). One-way ANOVA followed by Tukey post-hoc test. b) Top predicted upstream regulators by Ingenuity Pathway Analysis (IPA) from 158 DEGs (C13-treated vs. untreated, pAdj<0.05) identified by RNA-Seq studies from primary microglial cells stimulated with 1 μg/mL LPS for 5 h. The P-values of overlap are shown in parentheses. c) Assessment of gene expression levels for a set of key cytokines by qRT-PCR from WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 5 h (N=3). Data represent fold-changes relative to unstimulated WT cells (values were 1 or lower for unstimulated WT and MOK-KO controls; not shown). # P<0.05, ## P<0.01 and ### P<0.001 vs. unstimulated controls. One-way ANOVA followed by Tukey post-hoc test. d) Quantification of TNFα, IL-6, IFNβ and IL-1α levels by ELISA in supernatants from WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 5 h or overnight for IL-1α (N=5 for IL-6, TNFα and N=4 for IFNβ, IL-1α). One-way ANOVA followed by Tukey post-hoc test. e) Flow cytometry analysis IL-6 + cells in WT and MOK-KO SIM-A9 cells, previously stimulated with 1 μg/mL LPS for 5 h (left). Rescue of phenotype in MOK-KO cells resulting from cell transfection with FLAG-MOK-WT or FLAG-MOK-KD constructs and stimulation with 1 μg/mL LPS for 5 h (right) (N=3). One-way ANOVA followed by Tukey post-hoc test. f) Fold-changes of primary motor neurons (MNs) survival (relative to each control) after exposure to conditioned media from LPS-stimulated WT and MOK-KO cells (N=3). Student’s t test performed between shown groups, unpaired, two-tailed. Data in a, b, d-f are the mean -/+ S.E.M. from ‘N’ independent experiments, and *P<0.05, **P<0.01 and ***P<0.001. NS: not significant.

Article Snippet: MOK-knockout (MOK-KO) SIM-A9 cell clones (H14 and A7) were generated by CRISPR/Cas9 technology (Synthego, Redwood City, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: Data in a-e are from 3 independent experiments (N=3). a) Venn diagrams depicting the number of total (top) and protein-coding (bottom) DEGs between MOK-KO and WT SIM-A9 cells under basal conditions and/or after 1 μg/mL LPS stimulation for 5 h (pAdj. <0.05; log2 fold-change>2 and <-2); b) Heatmap showing unsupervised clustering analysis for the top 100 protein-coding genes based on relative expression levels in the four samples; c) GO ‘biological process’ term enrichment analysis of total DEGs for MOK-KO+LPS vs. WT+LPS. Shown are the top hits based on p-value (indicated in parenthesis) with at least three up/downregulated genes; d) Heatmap depicting clustering analysis for 100 coding DEGs identified for the MOK-KO+LPS vs. WT+LPS comparison (consisting of the top 80% downregulated and top 20% upregulated, genes to maintain proportionality of all significant DEGs found); e) Top-10 IPA-predicted upstream expression regulators from all coding DEGs comparing MOK-KO vs. WT cells upon LPS stimulation, indicating positive (activated) or negative (inhibited) z-scores. The results are from all genes that were found to be misregulated in the RNA-Seq deseq2 analysis in any comparison with pAdj of

Article Snippet: MOK-knockout (MOK-KO) SIM-A9 cell clones (H14 and A7) were generated by CRISPR/Cas9 technology (Synthego, Redwood City, CA, USA).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a,b) Quantification of nuclear pBrd4 levels by confocal IF with anti-pBrd4 (green) and DAPI staining of primary microglial cells pre-treated with 10 μM C13 (or DMSO) for 1 h and stimulated or not with 1 μg/mL LPS for 4 h. The data and images shown correspond to one experiment (N=20 analyzed images per condition) and are representative of two independent experiments. Scale bar: 10 μm. Student’s t-test (between two groups as indicated), unpaired, one-tailed. c,d) Quantification (c) and representative image (d) of Western blot analyses with anti-pBrd4 and anti-Brd4 (normalized to Brd4 or α-tubulin signal, respectively) in lysates of WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 1 h. Data are from three independent experiments (N=3). Student’s t-test, unpaired, one-tailed. e,f) Quantification (e) and representative images (f) of confocal IF analyses of nuclear pBrd4 levels with anti-pBrd4 (green) and DAPI staining of WT and MOK-KO SIM-A9 cells stimulated or not with 1 μg/mL LPS for 1 h. Signal was enhanced by 50% in all four images in the first row for better visualization. Images are from one out of three independent experiments (N=3). Scale bar: 10 μm. Student’s t-test, unpaired, two-tailed. g) ChIP-PCR assessment of Brd4-binding to specific cytokine promoters. Represented data are relative values (percentage of input) of PCR products for IL-6, IFNβ and TNFα promoters after ChIP assay with either anti-Brd4 or IgG control antibodies with chromatin isolated from WT or MOK-KO cells treated or not with 1 μg/mL of LPS for 1 hour (N=4). Student’s t-test, paired, one-tailed. Data are the mean -/+ S.E.M. from ‘N’ independent experiments, and *P<0.05, **P<0.01 and ***P<0.001. NS: not significant.

Article Snippet: MOK-knockout (MOK-KO) SIM-A9 cell clones (H14 and A7) were generated by CRISPR/Cas9 technology (Synthego, Redwood City, CA, USA).

Techniques: Staining, One-tailed Test, Western Blot, Two Tailed Test, Binding Assay, Isolation

Journal: Nutrients

Article Title: Fatty Acid-Derived N -acylethanolamines Dietary Supplementation Attenuates Neuroinflammation and Cognitive Impairment in LPS Murine Model

doi: 10.3390/nu14183879

Figure Lengend Snippet: Effects of LPS and DS treatment on microglial morphology, proinflammatory cytokines, and NO production. ( a ) Representative images of SIM-A9 microglia cell culture after LPS (1 μg/mL) and DS (0.1, 1 and 10 μg/mL) treatment, scale bar—100 μm. ( b ) Western blot analysis of SIM-A9 microglia cell lysates ( a ) INFγ (25 kDa), TNFα (26 kDa), IL-1β (30 kDa), IL6 (22 kDa), and MHCII (47 kDa) were detected in LPS-activated SIM-A9 cell lysates with β-tubulin protein expression used as a loading control. Original uncropped images of blots are shown in . ( c ) Relative quantity of INFγ, TNFα, IL-1β, IL6, and MHCII in SIM-A9 microglia cell lysates determined with Western blot, n = 4, * p < 0.001—compared to Veh, + p < 0.001—compared to LPS. ( d ) Nitrite production was detected with the Griess test, n = 6 per group. Data are presented as mean ± SEM, +++ p < 0.001 — compared to LPS, *** p < 0.001 — compared to Veh.

Article Snippet: For in vitro studies, we used a microglial cell line SIM-A9 (T0247-GVO-ABM, BioCat, Heidelberg, Germany).

Techniques: Cell Culture, Western Blot, Expressing

Journal: bioRxiv

Article Title: Phf15 —a novel transcriptional repressor regulating inflammation in mouse microglia

doi: 10.1101/2019.12.17.879940

Figure Lengend Snippet: (A) Percent reduction in Phf15 transcript expression in Phf15 knockout SIM-A9 microglia ( Phf15 KO, red bar) compared to control (Ctrl, open bar). 24-hour time course experiments showing relative mRNA expression levels of Tnfα (B), Il-1β (D), and Nos2 (F) after LPS stimulation. No stimulation (0 hr time point or baseline) expression of Tnfα (C), Il-1β (E) and Nos2 (G) are also shown. All data are mean ±SEM (n = 3 per condition). Unpaired t-tests between Phf15 KO and control cells for percent reduction and for individual timepoints: asterisks indicate * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. LPS, lipopolysaccharide; Tnfα , tumor necrosis factor alpha; inducible; Il-1β , Interleukin 1 beta; Nos2 , nitric oxide synthase.

Article Snippet: Viral supernatant was harvested after 48 hours and incubated with SIM-A9 murine microglial cells in SIM-A9 complete medium (DMEM/F12, Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Chicago, IL), 5% horse serum (HS; GE Healthcare Life Sciences, Chicago, IL), 1% Pen-Strep (Life Technologies, Carlsbad, CA)).

Techniques: Expressing, Knock-Out

Journal: bioRxiv

Article Title: Phf15 —a novel transcriptional repressor regulating inflammation in mouse microglia

doi: 10.1101/2019.12.17.879940

Figure Lengend Snippet: (A) Fold overexpression (OE) of Phf15 in SIM-A9 microglia (red bar) versus control cells (Ctrl, open bar). 24-hour time course experiments showing relative mRNA expression levels of Tnfα (B), Il-1β (D), and Nos2 (F) after LPS stimulation. Baseline (0 hour time point, No stimulation) expression of Tnfα (C), Il-1β (E), and Nos2 (G) are displayed separately from time course experiments. All data are mean ±SEM (n = 3 per condition). Unpaired t-tests between Phf15 OE and control cells for fold-overexpression and for individual time points: asterisks indicate * p <0.05, ** p <0.01. LPS, lipopolysaccharide; Tnfα , tumor necrosis factor alpha; inducible; Il-1β , Interleukin 1 beta; Nos2 , nitric oxide synthase.

Article Snippet: Viral supernatant was harvested after 48 hours and incubated with SIM-A9 murine microglial cells in SIM-A9 complete medium (DMEM/F12, Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Chicago, IL), 5% horse serum (HS; GE Healthcare Life Sciences, Chicago, IL), 1% Pen-Strep (Life Technologies, Carlsbad, CA)).

Techniques: Over Expression, Expressing

Journal: bioRxiv

Article Title: Phf15 —a novel transcriptional repressor regulating inflammation in mouse microglia

doi: 10.1101/2019.12.17.879940

Figure Lengend Snippet: (A) Volcano plot representing the RNA-seq results. Orange dots represent differentially expressed genes in Phf15 knockout microglia compared to control (upregulated genes at a cutoff of log2fold change > 1.5 and p adj < 0.01; downregulated genes at a cutoff of log2fold change < −1.5 and p adj < 0.05). (B) GO analysis for significantly upregulated genes showing biological process categories related to response to virus and inflammatory response. (C) Upregulated genes associated with response to virus and inflammatory response in the No stimulation (baseline) condition. Relative FPKM values were obtained by normalizing FPKM values of Phf15 knockout SIM-A9 microglia to control FPKM values for each gene (n = 3 per condition). Statistics are by DESeq2: asterisks indicate ** p <0.01, #p<0.0001. (D) Top 5 enriched transcription factor binding motifs for the set of upregulated genes in the No stimulation (baseline) condition.

Article Snippet: Viral supernatant was harvested after 48 hours and incubated with SIM-A9 murine microglial cells in SIM-A9 complete medium (DMEM/F12, Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Chicago, IL), 5% horse serum (HS; GE Healthcare Life Sciences, Chicago, IL), 1% Pen-Strep (Life Technologies, Carlsbad, CA)).

Techniques: RNA Sequencing Assay, Knock-Out, Binding Assay

Journal: bioRxiv

Article Title: Phf15 —a novel transcriptional repressor regulating inflammation in mouse microglia

doi: 10.1101/2019.12.17.879940

Figure Lengend Snippet: (A) Volcano plot representing the RNA-seq results. Orange dots represent differentially expressed genes in Phf15 knockout microglia 6 hours after LPS administration compared to control (upregulated genes at a cutoff of log2fold change > 1.5 and p adj < 0.01; downregulated genes at a cutoff of log2fold change < −1.5 and p adj < 0.05). (B) GO analysis for upregulated genes shows biological process categories associated with cytokine secretion and immunoregulatory interaction. (C) Upregulated genes associated with cytokine secretion and immunoregulatory interaction biological process categories 6 hours post LPS stimulation. Relative FPKM values were obtained by normalizing FPKM values of Phf15 knockout SIM-A9 microglia to control FPKM values for each gene (n = 3 wells per condition). Statistics are by DESeq2: asterisks indicate ** p <0.01, $p<0.001, #p<0.0001. (D) Transcription factor binding motifs for the set of upregulated genes 6 hours after LPS stimulation are enriched for Activator protein 1 (AP-1).

Article Snippet: Viral supernatant was harvested after 48 hours and incubated with SIM-A9 murine microglial cells in SIM-A9 complete medium (DMEM/F12, Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Chicago, IL), 5% horse serum (HS; GE Healthcare Life Sciences, Chicago, IL), 1% Pen-Strep (Life Technologies, Carlsbad, CA)).

Techniques: RNA Sequencing Assay, Knock-Out, Binding 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 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