Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: a – d Rat GMCs overexpressing the pcDNA3.1-ERK1/2, pcDNA3.1-SOX9, and pcDNA3.1-Cyclin D1 plasmids were cultured for 48 h. e – h GMCs were stimulated with sublytic C5b-9 for 24 h or 3 h after transfection with the shERK1/2, shSOX9, or shCyclin D1 plasmids. The changes in cellular proliferation were tested by CCK-8 ( a ) or EdU incorporation assays ( b , c ). The mRNA and protein expression levels of Cyclin D1, t-ERK1/2, and SOX9 were assessed by qRT-PCR ( d , e ) and IB ( f – h ), respectively. * p < 0.05, ** p < 0.01 versus the pcDNA3.1 group or the shCTR group; Δ p < 0.05, ΔΔ p < 0.01 versus the shCTR + sublytic C5b-9 group. Data were represented as the means ± SD ( n = 5 in each group for the CCK‐8 and EdU incorporation assays, n = 3 in each group in the other experiments).

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: Cell Culture, Transfection, CCK-8 Assay, Expressing, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: a Scheme showing full-length (FL) and the three indicated deletion mutants of the pGL-Cyclin D1 promoter according to JASPAR prediction. b – f Rat GMCs were cultured with various treatments or transfected with different plasmids with or without sublytic C5b-9 stimulation for 3 h. The activity of the Cyclin D1 promoter and the binding elements of SOX9 were measured by luciferase reporter assay ( b – d ) and ChIP-PCR ( e , f ). ** p < 0.01 versus the MEM, Thy-1 Ab, Thy-1 Ab + HIS, or Thy-1 Ab + C6DS group or versus the pcDNA3.1, shCTR, or pGL3-basic + sublytic C5b-9 group; ΔΔ p < 0.01 versus the shCTR + sublytic C5b-9, FL + sublytic C5b-9, -985 to +130 nt + sublytic C5b-9, or -582 to +130 nt + sublytic C5b-9 group. Data were represented as the means ± SD ( n = 3 in each group in all experiments).

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: Cell Culture, Transfection, Activity Assay, Binding Assay, Luciferase, Reporter Assay

Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: a – d Rat GMCs cotransfected with pcDNA-SOX9 and shERK1/2 plasmids were stimulated with sublytic C5b-9 for the indicated times. GMC proliferation was determined by CCK-8 ( a ) and EdU incorporation assays ( b ). Cyclin D1 promoter activity and protein level were analyzed by luciferase reporter assay ( c ) and IB ( d ), respectively. ** p < 0.01 versus the pcDNA3.1 + shCTR group; Δ p < 0.05, ΔΔ p < 0.01 versus the pcDNA3.1 + shCTR + sublytic C5b-9 group; # p < 0.05 or ## p < 0.01 versus the pcDNA3.1 + shERK1/2 + sublytic C5b-9 group. Data were represented as the means ± SD ( n = 5 in each group for the CCK‐8 and EdU incorporation assays, n = 3 in each group in the other experiments).

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: CCK-8 Assay, Activity Assay, Luciferase, Reporter Assay

Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: Rat GMCs pretransfected with shSOX9 plasmid were treated with TSA (20 μM) for 30 min, followed by sublytic C5b-9 stimulation for 24 h or 3 h. GMC proliferation was determined by CCK-8 ( a ) and EdU incorporation assays ( b ). Cyclin D1 promoter activity and occupation by SOX9 were analyzed by luciferase reporter assay ( c ) and ChIP-PCR ( d ). The levels of SOX9 acetylation and phosphorylation and the protein expression of Cyclin D1 were determined by co-IP and IB ( e ). ** p < 0.01 versus the shCTR + DMSO group; Δ p < 0.05 or ΔΔ p < 0.01 versus the shCTR + DMSO + sublytic C5b-9 group; ## p < 0.01 versus the shCTR + TSA + sublytic C5b-9 group. Data were represented as the means ± SD ( n = 5 in each group for CCK‐8 assays, n = 3 in each group in the other experiments).

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: Plasmid Preparation, CCK-8 Assay, Activity Assay, Luciferase, Reporter Assay, Phospho-proteomics, Expressing, Co-Immunoprecipitation Assay

Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: a – e Rat GMCs were transfected with HA-tagged pcDNA-SOX9 wild-type (WT) or different phospho-mutant pcDNA-SOX9 plasmids (S64A/E or S181A/E) in the presence or absence of sublytic C5b-9. HA-SOX9 protein was purified, and the SOX9 phosphorylation sites were identified by mass spectrometry (MS, a ). Cyclin D1 promoter activity and promoter occupation were determined by luciferase reporter ( b ) and ChIP-PCR assays ( c ). Cyclin D1 abundance and nuclear SOX9 levels were tested by IB ( d ). SOX9 acetylation and its association with KAT7, PCAF, and GCN5 were measured by co-IP assays ( e ). f Rat GMCs overexpressing the ERK1/2 gene were transfected with pcDNA-SOX9 WT or different phospho-mutant pcDNA-SOX9 plasmids for 48 h. The Cyclin D1 protein level was measured by IB. ** p < 0.01 versus the pcDNA3.1 or pcDNA-ERK1/2 + pcDNA3.1 group; Δ p < 0.05, ΔΔ p < 0.01 versus the WT or pcDNA-ERK1/2 + WT group. Data were represented as the means ± SD ( n = 3 in each group in all experiments).

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: Transfection, Mutagenesis, Purification, Phospho-proteomics, Mass Spectrometry, Activity Assay, Luciferase, Co-Immunoprecipitation Assay

Journal: Experimental & Molecular Medicine

Article Title: Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

doi: 10.1038/s12276-021-00589-9

Figure Lengend Snippet: a , b The protein levels of ERK1/2 (t-ERK1/2 and p-ERK1/2), SOX9 (SOX9 and p-SOX9), and Cyclin D1 ( a ) as well as SOX9 acetylation and its interaction with p-ERK1/2 ( b ) were measured at 3 h in the renal tissues of Thy-1N from different groups as indicated. c Renal ultrastructural changes on day 7 were examined by EM. d , e The changes in glomerular cell number in each group on day 7 were examined by H&E staining under LM. e The total contents of urinary protein (mg/24 h) on day 7 were detected. * p < 0.05, ** p < 0.01 versus the NRS group; Δ p < 0.05, ΔΔ p < 0.01 versus the LV-shCTR + Thy-1N group. Data were represented as the means ± SD ( n = 5 in vivo in each group). f A putative scheme for the molecular regulation of GMC proliferation triggered by sublytic C5b-9. In response to extracellular sublytic C5b-9 attack, ERK1/2 is phosphorylated and enters the GMC nucleus to form a complex with SOX9, leading to SOX9 phosphorylation at the Ser64 and Ser181 residues. Phosphorylation of SOX9 at these sites not only induces SOX9 nuclear localization but also enhances its acetylation by promoting the interaction of SOX9 with HATs, including KAT7, PCAF, and GCN5. In addition, ERK1/2-upregulated GCN5 is also implicated in SOX9 acetylation. Nuclear phosphorylated or acetylated SOX9 can be recruited to the Cyclin D1 promoter regions, causing its gene induction and GMC proliferation.

Article Snippet: pMT-ERK1, pcDNA-ERK2, and pcDNA-SOX9 (HA-tagged) plasmids were obtained from Addgene. pcDNA-Cyclin D1 was constructed by inserting the ORF of rat Cyclin D1 cDNA (NM_171992) into the pcDNA vector.

Techniques: Staining, In Vivo, Phospho-proteomics

Journal: Oncogenesis

Article Title: Selective repression of the oncogene cyclin D1 by the tumor suppressor miR-206 in cancers

doi: 10.1038/oncsis.2014.26

Figure Lengend Snippet: miR-206 directly targets CCND1 at a specific 3′-UTR site. ( a ) Schematic of putative miR-206 target site within the human CCND1 3′-UTR as predicted by TargetScan and PicTar algorithms. Mutations introduced into the full-length human CCND1 3′-UTR are highlighted in red. ( b ) Relative luciferase activity was measured in 3T3-L1 cells co-transfected with a luciferase reporter construct containing a fragment of the mouse Ccnd1 3′-UTR encompassing the miR-206 binding site or empty pMIR-Report vector, along with miR-ve or miR-206. Data represent relative Firefly values as determined by the ratio of Firefly to Renilla luciferase activity with control miR-ve set as 1. Values are the mean±s.e.m. of three separate experiments. ( c ) Relative luciferase activity was measured in H1299 cells co-transfected with reporter constructs containing human full-length wild-type (WT)- CCND1 3′-UTR or mutant (MUT)- CCND1 3′-UTR along with miR-ve or miR-206. Firefly luciferase units were normalized against Renilla luciferase units to control for transfection efficiency. Percentage Firefly luciferase values were determined with control miR-ve set at 100%. Data represent the mean+s.e.m. of three independent experiments. A paired t -test was used to determine significance between miR-ve and miR-206 transfected cells (* indicates P <0.05).

Article Snippet: Luciferase reporter plasmid containing the full-length human cyclin D1 3′-UTR was obtained from Switchgear Genomics (Carlsbad, CA, USA).

Techniques: Luciferase, Activity Assay, Transfection, Construct, Binding Assay, Plasmid Preparation, Control, Mutagenesis