pcaggs drr mcherry donor ef1a bfp  (Addgene inc)

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pY641) and γH2AX in thioglycolate-elicited peritoneal macrophages (Thio-PM) with Etoposide (Eto) and IL-4 treatment. The right panel shows the relative intensity of pSTAT6 (pY641) ( n = 4 independent experiments). B Immunofluorescence images of staining (pSTAT6 (pY641), red; DAPI, blue) of IL-4-stimulated Thio-PMs treated with Eto. Scale bars 50 μm. Right panel shows the quantification of pSTAT6 (pY641) ( n = 3 biological replicates per group). C Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Thio-PMs with different inhibitors. KU55933, an inhibitor of ATM. Berzosertib, inhibitor of ATR. NU7026, inhibitor of DNA-PK. AZD7762, inhibitor of CHK1/2. The right panel shows the quantification of pSTAT6 (pY641) ( n = 3 independent experiments). D Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with indicated sgRNA. The right panel shows the quantification of pSTAT6 (pY641)/tSTAT6 ( n = 3 independent experiments). E Dual luciferase reporter of STAT6 in RAW 264.7 cells treated with Eto and NU7026 ( n = 3 per group). Data are mean ± s.e.m. p-value was calculated by paired two-tailed Student’s t test ( A , D ), unpaired two-tailed Student’s t test ( B ), one-way ANOVA with Dunnett’s correction ( C , E ).

Article Snippet: Dual luciferase reporter of STAT6 (4 × S6 reporter) (#35554) and DNA repair reporter plasmids (#98896, #98895, #26477) were obtained from Addgene.

Techniques: Western Blot, Immunofluorescence, Staining, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Co-IP of STAT6-Turbo and JAK1, DNA-PK, ATM, and ATR in RAW 264.7 cells with TurboID. B Proximity ligation assay (PLA) of STAT6 and DNA-PK in Thio-PMs treated with Eto. Scale bars 10 μm. C The quantification of PLA signal in ( B ) ( n = 3 biological replicates per group, with more than 100 cells). D Diagram of phosphorylated sites in mouse STAT6 after Eto treatment. E Immunoblots of pSTAT6 (pY641) and tSTAT6 in Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, S807A, S807E). F Immunoblots of pSTAT6 (pY641) in STAT6-mutants-expressed Cas9 tg+ Thio-PMs with Prkdc sgRNA under IL-4 & Eto co-treatment. G Dual luciferase reporter of STAT6 in RAW 264.7 cells expressing STAT6 mutants under IL-4 treatment ( n = 3 biological replicates per group). H Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg+ Thio-PMs with Prkdc sgRNAs. I Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with Ku70 or Ku80 sgRNA. J Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Thio-PMs pre-treated with NU7026. The right panel shows the relative intensity of pSTAT6 (pS807) ( n = 3 independent experiments). K Immunoblots of pSTAT6 (pS807) and pSTAT6 (pY641) in Thio-PMs with indicated treatment. AS1517499, an inhibitor of STAT6 (block of pY641). L Immunoblots of pSTAT6 (pS807) and tSTAT6 in Eto-treated Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, Y641F). The blot results are representative of three biologically independent repeats in ( A , E , F , H , I , K and L ). Data are mean ± s.e.m. Unpaired two-tailed Welch’s t test ( C ), One-way ANOVA with Dunnett’s correction ( G ), paired two-tailed Student’s t test ( J ).

Article Snippet: Dual luciferase reporter of STAT6 (4 × S6 reporter) (#35554) and DNA repair reporter plasmids (#98896, #98895, #26477) were obtained from Addgene.

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Western Blot, Expressing, Luciferase, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Principal component (PC) analysis plot of Thio-PMs from Stat6 WT/WT , Stat6 S807A/S807A and Stat6 S807E/S807E mice, calculated by DESeq2. B Gene set enrichment analysis (GSEA) in pathways “Recatome_DNA_Repair”, “Hallmark_Inflammatory_response” and “SAUL_SEN_MAYO”, compared Stat6 S807E/S807E with Stat6 S807A/S807A Thio-PMs, calculated by GSEA (v4.3.2). C , D DNA repair capacity of Thio-PMs from Stat6 WT/- , Stat6 S807A/- and Stat6 S807E/- mice. The representative images of immunofluorescence staining (γH2AX, red; DAPI, blue) of PMs ( C ), and the quantifications (γH2AX positive proportions and mean fluorescence intensity (MFI)) are showed in ( D ) ( n = 3 biological replicates per group). Scale bars 100 μm. E STAT6 CUT&Tag binding enrichment of Thio-PMs from Stat6 WT/- , Stat6 S807A/- and Stat6 S807E/- mice after IL-4 treatment ( n = 3 biological replicates per group), which was assessed by qPCR. B1/B2/B3, three STAT6-binding sties around Brca1 locus. U, three STAT6-binding sties around the Ube2t locus. F Diagram of transcription co-factors screening. TFs regulating Brca1 or Ube2t were predicted by AnimalTFDB 4.0, and their expression was analyzed by RNA-seq (TPM > 0.5). G Endogenous Co-IP of STAT6 and PU.1 in Thio-PMs co-treated with Eto and IL-4 for 2 h. H PLA of STAT6 and PU.1 in Thio-PMs co-treated with Eto and IL-4 for 2 h. The right panel shows the quantification of the PLA signal ( n = 4 biological replicates per group). Scale bars 5 μm. I PU.1 CUT&Tag binding enrichment of Thio-PMs from Stat6 WT/- , Stat6 S807A/- and Stat6 S807E/- mice after IL-4 treatment ( n = 3 biological replicates per group), which was assessed by qPCR. B-1/B-2, two PU.1-binding sties around Brca1 locus. U-1/U-2/U-3, three PU.1-binding sties around the Ube2t locus. J Immunoblots of BRCA1 and UBE2T in Thio-PMs from Stat6 WT/WT , Stat6 S807A/S807A and Stat6 S807E/S807E mice with indicated treatments. DB2313, an inhibitor of PU.1. K Immunoblots of γH2AX in Thio-PMs from Stat6 WT/- , Stat6 S807A/- and Stat6 S807E/- mice with the indicated repair period. The blot data are representative of three biologically independent repeats ( G , J and K ). Data are mean ± s.e.m. The p -value was calculated by one-way ANOVA with Dunnett’s correction.

Article Snippet: Dual luciferase reporter of STAT6 (4 × S6 reporter) (#35554) and DNA repair reporter plasmids (#98896, #98895, #26477) were obtained from Addgene.

Techniques: Immunofluorescence, Staining, Fluorescence, Binding Assay, Expressing, RNA Sequencing, Co-Immunoprecipitation Assay, Western Blot

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pS817) and γH2AX in hMDMs under indicated treatment. B Quantification of pSTAT6 (pS817)/α-Tubulin in ( A ) ( n = 5 biological replicates per group). C mRNA expression of BRCA1 and UBE2T in hMDMs expressing hSTAT6 mutants (WT, S817A, S817E) after 16-h hIL-4 treatment ( n = 5 biological replicates per group). D DNA repair capacity of THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E). The right panel shows the quantification ( n = 3 independent experiments). E mRNA expression of P16-INK4A and P21-CIP1 in THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E) under Eto-induced senescence ( n = 3 biological replicates per group). F Phagocytosis assay of THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E). All groups had 30-min phagocytosis period. The right panel shows the quantification ( n = 4 biological replicates per group). G Representative image of lung sections from control and COPD donors. Scale bar 100 μm. H Quantification of P16 + cells in lungs from control and COPD donors ( n = 6 biological replicates per group). I Quantification of P16 + macrophages (CD68 + ) in lungs from control and COPD donors ( n = 6 biological replicates per group). J Immunoblots of pSTAT6 (pS817), P16-INK4A and γH2AX in lungs from control, smoker, and COPD donors ( n = 4 biological replicates per group). K Quantification of pSTAT6 (pS817)/tSTAT6 in lungs from control, smoker, and COPD donors ( n = 4 biological replicates per group). L Spearman’s correlation between pSTAT6 (pS817)/tSTAT6 and γH2AX in human lungs from control ( n = 10), smoker ( n = 4), and COPD donors ( n = 10). M Spearman’s correlation between pSTAT6 (pS817)/tSTAT6 and P16-INK4A in human lungs from control ( n = 10), smoker ( n = 4), and COPD donors ( n = 10). Data are mean ± s.e.m. p -value was calculated by paired two-tailed Student’s t test ( B , C and D ), one-way ANOVA with Dunnett’s correction ( E , F and K ), unpaired two-tailed Student’s t test ( H and I ), two-tailed Spearman’s correlation coefficient analysis ( L and M ).

Article Snippet: Dual luciferase reporter of STAT6 (4 × S6 reporter) (#35554) and DNA repair reporter plasmids (#98896, #98895, #26477) were obtained from Addgene.

Techniques: Western Blot, Expressing, Phagocytosis Assay, Control, Two Tailed Test