Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: Runx2 activation boosted CDK8 transcription in BEAS-2B cells. A The downstream target genes of Runx2 were predicted through CHEA, hTFTarget, and KnockTF2.0. B The effects of acute CSE incubation on the top 3 target genes were evaluated in BEAS-2B cells by RT-PCR. C SiRunx2 plasmids were established and transfected to repress Runx2 expression in BEAS-2B cells. Then, the influences of siRunx2 on CSE-induced the changes of the top 3 target genes were estimated using RT-PCR. D , E CDK8 nuclear protein was measured in mouse lungs after CS exposure using western blotting ( D ) and densitometry analysis ( E ). F , G The effect of acute CSE on CDK8 nuclear protein was determined in BEAS-2B cells using western blotting ( F ) and densitometry analysis ( G ). H - K The effects of siRunx2 plasmids transfection on CSE-activated CDK8 and inflammatory cytokines were explored in BEAS-2B cells. H , I The proteins expression of Runx2 and CDK8 were detected using western blotting ( H ) and densitometry analyses ( I ). J The levels of inflammatory cytokines were measured using RT-PCR. K The effects of CDK8 inhibition on CSE-upregulated inflammatory cytokines were evaluated by RT-PCR. L , M Potential binding sequences for Runx2 were predicted in the CDK8 promoter ( L ), Runx2 andluciferase reporter plasmids were transfected and luciferase activity was detected ( M ). All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Activation Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Western Blot, Inhibition, Binding Assay, Luciferase, Activity Assay

Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: CS exposure induced Runx2 acetylation through repressing Sirt3. A - L The effects of acute CSE on acetylation and Sirts family were analyzed in BEAS-2B cells, as well as the levels of acetylation in animal models exposed to CS and in patients with COPD. The overall acetylation levels were observed in BEAS-2B cells exposed to acute CSE ( A , B ), COPD-like animal models exposed to CS ( C , D ), and patients with COPD ( E , F ) using western blotting. ( G , H ) The effects of acute CSE exposure on Sirts family were evaluated by western blotting ( G ) and densitometry analyses ( H ). I , J Sirt3 nuclear protein was determined in CSE-exposed BEAS-2B cells using western blotting ( I ) and densitometry analysis ( J ). K , L The effect of CS exposure on Sirt3 nuclear translocation was analyzed through IHC in lung tissues of COPD mice ( K ) and quantitative analysis ( L ). M , N The colocalization between Sirt3 and Runx2 was detected in mouse lungs using IF ( M ) and Mander colocalization coefficient was analyzed ( N ). O , P The impact of CSE exposure on Runx2 acetylation was determined by western blotting ( O ) and densitometry analysis ( P ). Q Molecular docking of Runx2 with Sirt3 was determined via the HDOCK method. R , S The impact of Sirt3 decrease on CSE-elevated Runx2 protein stability was analyzed by western blotting ( R ) and densitometry analysis ( S ). T , U The effect of Sirt3 decline on CSE-upregulated Runx2/CDK8 was estimated via western blotting ( T ) and densitometry analyses ( U ). V - Y The effect of Sirt3 downregulation on CSE-mediated Runx2 acetylation was explored with Co-IP. V IP: Runx2; Western blotting: Sirt3. W Densitometry analyses. X IP: Sirt3; Western blotting: Runx2. Y Densitometry analyses. All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Western Blot, Translocation Assay, Co-Immunoprecipitation Assay

Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: NR supplementation alleviated CS-activated Runx2/CDK8 axis in mouse lungs. A - R The impact of NR supplementation on CS-activated Runx2/CDK8 axis activation was explored in mouse lungs. A The content of NAD+ was detected in BEAS-2B cells after CSE treatment. B The effect of NR supplementation on CSE-downregulated NAD+ was evaluated in BEAS-2B cells. C , D The effect of NR supplementation on CSE-activated Runx2/CDK8 axis was estimated in BEAS-2B cells by western blotting ( C ) and densitometry analyses ( D ). E The concentration of NAD + was analyzed in mouse lungs after CS exposure. F The influence of NR pretreatment on CSE-evoked downregulation of NAD + was evaluated in mouse lungs. G , H The effect of NR supplementation on CS-activated Runx2/CDK8 axis was explored in mouse lungs via western blotting ( G ) and densitometry analyses ( H ). I , J Runx2-positive nuclei was determined in mouse lungs by IHC ( G ) and quantitative analysis ( H ). K , L ) Sirt3-positive nuclei was assessed in mouse lungs through IHC ( I ) and quantitative analysis ( J ). M - P The effects of NR supplementation on CS-promoted the production of inflammatory cytokines were estimated in mouse lungs via RT-PCR, including Cxcl1 ( M ), Cxcl2 ( N ), Il-6 ( O ), Cxcl15 ( P ). All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Activation Assay, Western Blot, Concentration Assay, Reverse Transcription Polymerase Chain Reaction