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: bioRxiv

Article Title: Base Editing as a Therapeutic Strategy for Autosomal Dominant Tubulointerstitial Kidney Disease caused by Pathogenic UMOD Variants

doi: 10.64898/2026.10.15.758759

Figure Lengend Snippet: CRISPRa-mediated activation of endogenous UMOD enables functional testing of knockdown strategies. ( A ) Schematic of the synergistic activation mediator (SAM) system for CRISPR-mediated transcriptional activation. MS2-containing sgRNAs target the proximal UMOD promoter and recruit dCas9-VP64 together with the MS2-p65-HSF1 (MPH) helper complex. ( B ) Positions of the ten sgRNAs (act_sgRNA1–10) targeting the proximal UMOD promoter. Guides target both strands (arrows) within −20 to −239 bp upstream of the transcription start site (TSS), located within exon 1. ( C ) UMOD expression quantified by ΔΔCt qRT-PCR in CRISPRa-competent HEK293T cells (HEK293T/ dCas9-VP64 + MPH) after transfection with each individual sgRNA, with a combination of all sgRNAs (act_all) or with a combination of the three best-performing guides (act_sgRNA mix). CTL, mock transfection with non-targeting sgRNAs. Data are shown as mean ± SD from n = 3 replicates; experiment was independently repeated (n=2). ( D ) Left: schematic of hUREC isolation from human urine. Right: western blot of hUREC lysates after transfection with act_sgRNA mix or non-targeting sgRNAs, from three activated samples. Recombinant Uromodulin (0.25, 1 and 2.5 fmol) was loaded on the same blot for quantitative reference. β-actin served as loading control. ( E ) Deglycosylation analysis of CRISPRa-activated hURECs. Lysates were mock-treated or digested with Endo H or PNGase F. β-actin served as loading control. ( F ) Schematic of the experimental workflow: 1. base editing for splice-site disruption or stop codon introduction, 2. FACS enrichment of edited cells, 3. CRISPRa activation, and 4. analysis by qRT-PCR or western blot. (G) UMOD mRNA levels quantified by qRT-PCR in CRISPRa-activated HEK293T cells following splice-site disruption with seven sgRNA–base editor combinations (SSD_sgRNA1, 2, 4, 5, 6, 7 and 8 with ABE8.20m or BE4max), compared with base editor-only (no_sgRNA_CTL) controls (n = 4 biological replicates). ( H ) Left: Western blot of uromodulin in CRISPRa-activated HEK293T cells following stop codon introduction with STOP_sgRNA1, 3 and 4 and TadCBEd-V106W, compared with base editor-only transfected controls. Right: quantification of uromodulin protein levels (n = 3 biological replicates). β-actin served as loading control. ( G, H ) Data are shown as mean ± SD. Statistical significance was determined using ordinary one-way ANOVA followed by multiple comparisons testing. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: A total of 5 × 10 5 cells were electroporated per sample with 5μg plasmid DNA comprising dCas9-VP64 (Addgene plasmid #61422), MS2-p65-HSF1 (Addgene plasmid #61423), and the act_sgRNA mix at a 1:1:1 mass ratio.

Techniques: Activation Assay, Functional Assay, Knockdown, CRISPR, Expressing, Quantitative RT-PCR, Transfection, Isolation, Western Blot, Recombinant, Control, Analysis, Disruption, FACS

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