Journal: Advanced Science

Article Title: Astrocyte‐Associated ABCA1/APOE Responses Accompany Hippocampal Lipid Remodeling and Ectopic Neuronal Accumulation During Epileptogenesis

doi: 10.1002/advs.78329

Figure Lengend Snippet: Multi‐omics profiling reveals bexarotene‐associated metabolic and lipidomic remodeling, alterations in lipid‐handling proteins and MAPK phosphorylation, and reduced neuronal lipid droplet area following KA‐induced SE. (A,C) Volcano plots showing differentially abundant metabolites (A) and lipid species (C) in hippocampal samples from KA2W‐Bex and KA2W‐Veh mice in POS. Red and blue dots indicate higher‐ and lower‐abundance features, respectively, in the Bex group relative to the Veh group; dot size indicates the VIP score. (B) KEGG over‐representation analysis of downregulated metabolites in KA2W‐Bex vs. KA2W‐Veh hippocampi. Upregulated and downregulated metabolites were analyzed separately; the upregulated set was not displayed because only five metabolites had KEGG annotations, providing too few annotated features for an informative pathway analysis. Dot size indicates the number of mapped metabolites. Color encodes −log 10 ( q ), where q is the Benjamini–Hochberg‐adjusted p value; q values are labeled beside the points. (D) KEGG over‐representation analyses of upregulated and downregulated lipid species in KA2W‐Bex vs. KA2W‐Veh hippocampi. Dot size indicates the number of mapped lipid species. Color encodes −log 10 ( q ), where q is the Benjamini–Hochberg‐adjusted p value; q values are labeled beside the points. (E) Lollipop plots showing the top 20 lipid species with the largest absolute log2 fold change in each direction. Red and blue indicate increased and decreased lipid species, respectively, in the Bex group; dot size represents the VIP score. (F–I) Western blot analysis (F) and quantification of ABCA1 (G), APOE (H) and MERTK protein levels normalized to β‐Tubulin in KA‐induced mice with/without Bex treatment at 2 and 8 weeks post‐SE. (J,K) Western blot analysis (J) and quantification of phosphorylated p38 (p‐p38) relative to total p38 protein levels (K), normalized to β‐Tubulin, in KA‐induced mice with or without Bex treatment at 2 and 8 weeks post‐SE. (L,M) Western blot analysis (L) and quantification of phosphorylated Erk1/2 (p‐Erk1/2) relative to total Erk1/2 protein levels (M), normalized to β‐Tubulin, in KA‐induced mice with or without Bex treatment at 2 and 8 weeks post‐SE. (N,O) Quantification of the mean total LD area per TEM image in neuronal (N) and astrocytic (O) within the hippocampal hilus of KA2W‐Veh and KA2W‐Bex groups. (P,Q) Representative TEM images of neurons (P) and astrocytes (Q) with corresponding magnified views. Yellow dashed circles indicate the regions shown at higher magnification, and yellow arrowheads indicate LDs. Scale bars: 5 µm (main panels), 500 nm (magnified insets). Data are presented as mean ± SD. Panels A‐E, G–I and K–M, n = 3 mice per group. Panels N and O, n = 4 mice per group. Protein‐expression data (G–I, K, and M) were analyzed by two‐way ANOVA followed by Sidak's multiple‐comparisons test. TEM data (N,O) were analyzed using the two‐sided Mann–Whitney U test. * p < 0.05 and ** p < 0.01. Numbers above bars indicate exact non‐significant P values. For panels A–E and N–Q, data were obtained from the early post‐SE intervention cohort. Mice received Bex or matched Veh once daily for 7 consecutive days beginning 1 week after KA‐induced SE, and hippocampal tissues were collected at 2 weeks post‐SE. POS, positive‐ion mode; Veh, vehicle; Bex, bexarotene.

Article Snippet: The primary antibodies used were as follows: anti‐ABCA1 (NB100‐2068, 1:1000, Novus Biologicals), anti‐APOE (83728‐4‐RR, 1:1000, Proteintech), anti‐MERTK (ab95925, 1:1000, Abcam), anti‐phospho‐p38 (28796‐1‐AP, 1:1000, Proteintech), anti‐p38 (66234‐1‐Ig, 1:1000, Proteintech), anti‐phospho‐Erk1/2 (28733‐1‐AP, 1:1000, Proteintech), anti‐Erk1/2 (11257‐1‐AP, 1:1000, Proteintech), anti‐GFAP (ab4674, 1:5000, Abcam), anti‐β‐tubulin (BF9210, 1:5000, Affinity Biosciences), and anti‐GAPDH (HY‐P80954A, 1:5000, MedChemExpress).

Techniques: Biomarker Discovery, Profiling, Phospho-proteomics, Analysis, Labeling, Western Blot, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) KEGG enrichment analysis of down-regulated DEGs in Sik2 F/F ; LysM Cre cardiac macrophages on day 3 post-MI. The top 20 enriched pathways are listed. ( B ) DEGs (fold change ≥ 2 and P .adjust ≤ 0.05) overlapped in top 20 of enriched pathways. ( C ) Transcription factor prediction was performed using TRRUST databases. The top 10 transcription factors are listed. ( D ) Immunoblotting analysis and quantification of P65 in the cytoplasm and nucleus of WT and SIK2-knockout PMs and treated with TNF-α (20 ng/ml) or vehicle ( n = 4) for 1 hour. ( E ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression in WT and SIK2-knockout PMs stimulated with TNF-α for 24 hours ( n = 6). ( F ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression ( n = 6). PMs were pretreated with ARN3236 (3 μmol) and stimulated with TNF-α for 24 hours. ( G ) WikiPathways enrichment analysis of differentially phosphorylated proteins in PMs treated with ARN3236 (3 μmol) or control. The top 20 enriched pathways are listed. ( H ) Immunoblotting analysis and quantification of phosphorylated P65 (p-P65), P65, phosphorylated P38 (p-P38), and P38 expression in WT and SIK2-knockout PMs stimulated with TNF-α or vehicle ( n = 6). ( I and J ) Flow cytometry analysis of efferocytosis in WT or LeSik2 Raw264.7 cells [stimulated with SB 203580 (10 μmol) or not] treated with CFSE-prelabeled RBCs ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Analysis, Western Blot, Knock-Out, Expressing, Control, Flow Cytometry, Significance Assay

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Venn diagram delineating the intersection of SIK2-binding protein candidates in the top enriched pathways identified in , that is, NF-κB signaling pathway and MAPK signaling pathway. LC-MS/MS, liquid chromatography–tandem mass spectrometry. ( B ) Top proteins interacting with SIK2 identified through mass spectrometry analysis of peptides pulled down using anti-Flag antibody in HEK293T cells transfected with Flag-Sik2. ( C ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 using anti-Flag antibody, followed by immunoblotting with corresponding antibodies. HA, hemagglutinin. ( D and E ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 and HA-Traf2 using anti-Flag (D) or anti-HA (E) antibody, followed by immunoblotting with corresponding antibodies. FL, full length. ( F ) Immunoprecipitation (IP) in HEK293T cells cotransfected with HA-Traf2 and truncated Flag-Sik2 using an anti-HA antibody, followed by immunoblotting (IB) with corresponding antibodies. ( G ) Immunoprecipitation in HEK293T cells cotransfected with Flag-Sik2 and truncated HA-Traf2 using an anti-HA antibody, followed by immunoblotting with corresponding antibodies. ( H ) Western blotting analysis and quantification of SIK2, TRAF2, p-P38, P38, p-P65, and P65 expression in HEK293T cells transfected with SIK2 and si-TRAF2 or control ( n = 4). ( I ) Coimmunoprecipitation (Co-IP) analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and His-Ub, along with Flag-Sik2 or Flag-Sik2 T175A mutant. ( J ) Co-IP analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and Flag-Sik2, along with Myc-K48-Ub or Myc-K63-Ub. ( K ) Co-IP analysis of TRAF2 phosphate serine (p-Ser) and phosphate threonine (p-Thr) in HEK293T cells transfected with HA-Traf2, along with Flag-Sik2 or Flag-Sik2 T175A mutant.

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Analysis, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Mutagenesis, Tandem Mass Spectroscopy

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Experimental design depicting AAV and P38 inhibitor SB 203580 injection in mice before or after MI. ip, intraperitoneally. ( B ) Immunoblotting analysis and quantification of SIK2 in infarct macrophages from AAV.F4/80.SIK2 or AAV.F4/80.NC injected mice ( n = 4 for AAV.F4/80.NC; n = 6 for AAV.F4/80.SIK2). ( C ) Representative parasternal long-axis views and M-mode images are shown. Summarized data of echocardiographic measurements in WT mice administrated with AAV.F4/80.SIK2 or AAV.F4/80.NC treated with SB 203580 or vehicle ( n = 6). ( D ) Masson’s trichrome staining for infarct size measurements in designated groups at day 14 after MI ( n = 6). ( E ) Representative parasternal long-axis views and M-mode images are shown. ( F and G ) Summarized data of echocardiographic measurements in WT mice administrated with AAV.F4/80.SIK2 T175A or AAV.F4/80.NC ( n = 6). ( H ) Masson’s trichrome staining for infarct measurements in designated groups at day 14 after MI ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Injection, Western Blot, Analysis, Staining

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) KEGG enrichment analysis of down-regulated DEGs in Sik2 F/F ; LysM Cre cardiac macrophages on day 3 post-MI. The top 20 enriched pathways are listed. ( B ) DEGs (fold change ≥ 2 and P .adjust ≤ 0.05) overlapped in top 20 of enriched pathways. ( C ) Transcription factor prediction was performed using TRRUST databases. The top 10 transcription factors are listed. ( D ) Immunoblotting analysis and quantification of P65 in the cytoplasm and nucleus of WT and SIK2-knockout PMs and treated with TNF-α (20 ng/ml) or vehicle ( n = 4) for 1 hour. ( E ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression in WT and SIK2-knockout PMs stimulated with TNF-α for 24 hours ( n = 6). ( F ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression ( n = 6). PMs were pretreated with ARN3236 (3 μmol) and stimulated with TNF-α for 24 hours. ( G ) WikiPathways enrichment analysis of differentially phosphorylated proteins in PMs treated with ARN3236 (3 μmol) or control. The top 20 enriched pathways are listed. ( H ) Immunoblotting analysis and quantification of phosphorylated P65 (p-P65), P65, phosphorylated P38 (p-P38), and P38 expression in WT and SIK2-knockout PMs stimulated with TNF-α or vehicle ( n = 6). ( I and J ) Flow cytometry analysis of efferocytosis in WT or LeSik2 Raw264.7 cells [stimulated with SB 203580 (10 μmol) or not] treated with CFSE-prelabeled RBCs ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Analysis, Western Blot, Knock-Out, Expressing, Control, Flow Cytometry, Significance Assay

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Venn diagram delineating the intersection of SIK2-binding protein candidates in the top enriched pathways identified in , that is, NF-κB signaling pathway and MAPK signaling pathway. LC-MS/MS, liquid chromatography–tandem mass spectrometry. ( B ) Top proteins interacting with SIK2 identified through mass spectrometry analysis of peptides pulled down using anti-Flag antibody in HEK293T cells transfected with Flag-Sik2. ( C ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 using anti-Flag antibody, followed by immunoblotting with corresponding antibodies. HA, hemagglutinin. ( D and E ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 and HA-Traf2 using anti-Flag (D) or anti-HA (E) antibody, followed by immunoblotting with corresponding antibodies. FL, full length. ( F ) Immunoprecipitation (IP) in HEK293T cells cotransfected with HA-Traf2 and truncated Flag-Sik2 using an anti-HA antibody, followed by immunoblotting (IB) with corresponding antibodies. ( G ) Immunoprecipitation in HEK293T cells cotransfected with Flag-Sik2 and truncated HA-Traf2 using an anti-HA antibody, followed by immunoblotting with corresponding antibodies. ( H ) Western blotting analysis and quantification of SIK2, TRAF2, p-P38, P38, p-P65, and P65 expression in HEK293T cells transfected with SIK2 and si-TRAF2 or control ( n = 4). ( I ) Coimmunoprecipitation (Co-IP) analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and His-Ub, along with Flag-Sik2 or Flag-Sik2 T175A mutant. ( J ) Co-IP analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and Flag-Sik2, along with Myc-K48-Ub or Myc-K63-Ub. ( K ) Co-IP analysis of TRAF2 phosphate serine (p-Ser) and phosphate threonine (p-Thr) in HEK293T cells transfected with HA-Traf2, along with Flag-Sik2 or Flag-Sik2 T175A mutant.

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Analysis, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Mutagenesis, Tandem Mass Spectroscopy

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Experimental design depicting AAV and P38 inhibitor SB 203580 injection in mice before or after MI. ip, intraperitoneally. ( B ) Immunoblotting analysis and quantification of SIK2 in infarct macrophages from AAV.F4/80.SIK2 or AAV.F4/80.NC injected mice ( n = 4 for AAV.F4/80.NC; n = 6 for AAV.F4/80.SIK2). ( C ) Representative parasternal long-axis views and M-mode images are shown. Summarized data of echocardiographic measurements in WT mice administrated with AAV.F4/80.SIK2 or AAV.F4/80.NC treated with SB 203580 or vehicle ( n = 6). ( D ) Masson’s trichrome staining for infarct size measurements in designated groups at day 14 after MI ( n = 6). ( E ) Representative parasternal long-axis views and M-mode images are shown. ( F and G ) Summarized data of echocardiographic measurements in WT mice administrated with AAV.F4/80.SIK2 T175A or AAV.F4/80.NC ( n = 6). ( H ) Masson’s trichrome staining for infarct measurements in designated groups at day 14 after MI ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Injection, Western Blot, Analysis, Staining