Journal: Scientific Reports

Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

doi: 10.1038/s41598-019-38809-y

Figure Lengend Snippet: The expression and methylation status of Ctse gene in CD4+ T cells isolated from MRL/lpr lupus-prone (MRL) and C57BL/6 (B6) mice. ( A ) mRNA expression of Ctse in CD4+, CD8+, B cells and macrophages in B6 and MRL mice. *** p < 0.001. ( B ) Western blot analyses of CTSE in CD4+, CD8+, B cells and macrophages in B6, MRL and mice. ( C ) Methylated region expanding 583 bp in Ctse intron 1 in B6 mice demonstrated by UCSC Genome Browser. In MRL mice, 583 bp region is hypomethylated. Duplicate reads are indicated by arrows. ( D ) Nucleotide sequence within 583 bp flanking region of Ctse intron 1 in B6 mice. Among 13 CpGs (red and underlined), 11th CpG (boxed) includes mCGCG binding motif for Kaiso. After genomic DNAs were bisulphite converted, amplified by PCR using Primer F1 and R1, subcloned, and isolated clones sequenced. 98.6% of CpGs (71 out of 72 colonies) in B6 mice and 76.9% CpGs (50 of 65) in MRL mice were methylated. ( E ) The structure of Kaiso, SMRT (Silencing Mediator of Retinoic acid and Thyroid hormone receptors)/N-CoR (Nuclear hormone receptor Co-Repressor), and HDAC3 (Histone deacetylase 3) complex. BTB (for BR-C, ttk and bab), POZ (for Pox virus and Zinc finger) and C2H2 zinc-finger domains are shown. ( F ) DNA sequencing of bisulphite converted DNAs in Ctse intron 1 in B6 and MRL mice. Percentage of methylation of 11th CpG is shown (n = 5). ***p < 0.001.

Article Snippet: CD4+ T lymphocytes were purified using the RosetteSep Human CD4+ TCD4+ T Cell Enrichment Cocktail (Stem Cell Technologies).

Techniques: Expressing, Methylation, Isolation, Western Blot, Sequencing, Binding Assay, Amplification, Clone Assay, Histone Deacetylase Assay, Virus, DNA Sequencing

Journal: Scientific Reports

Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

doi: 10.1038/s41598-019-38809-y

Figure Lengend Snippet: Kaiso binding to mCGCG motif located in Ctse intron 1 and HDAC3 (Histone deacetylase 3) in CD4+ T cells from MRL mice. ( A ) Methylation specific PCR analysis in EL4 cells treated with or without 1 μM of 5-azaC, and CD4+ T cells derived from B6 and MRL mice. Methylation-sensitive restriction enzyme Acc II digests unmethylated CGCG motifs, while methylated CGCG is resistant to Acc II. The genomic DNAs were digested with Acc II, amplified by PCR using primer sets; Primer F2/R2 and Primer F2/R3. The densitometry intensity ratios of PCR products (F2R2/F2R3) are shown. *p < 0.05. ( B ) mRNA expression of Ctse in EL4 cells treated with 1 μM of 5-azacytidine (5-azaC) and/or 100 nM of Trichostatin A (TSA). *p < 0.05, **p < 0.01, ***p < 0.001. ( C ) DNA binding of Kaiso and HDAC3 around mCGCG motif located in Ctse intron 1 examined using ChIP-PCR in EL4 cells. EL4 cells treated with 1 μM of 5-azacytidine (5-azaC) and/or 30 nM of Trichostatin A (TSA). ChIP-PCR was performed in 3 independent culture experiments. **p < 0.01. ( D ) DNA binding of Kaiso and HDAC3 around mCGCG motif in CD4+ T cells isolated from B6 and MRL mice. The amount of precipitated DNAs was quantified using real-time PCR with primers specific to 583 bp region in Ctse intron 1. ChIP-PCR was performed in 3 independent animals. *p < 0.05.

Article Snippet: CD4+ T lymphocytes were purified using the RosetteSep Human CD4+ TCD4+ T Cell Enrichment Cocktail (Stem Cell Technologies).

Techniques: Binding Assay, Histone Deacetylase Assay, Methylation, Derivative Assay, Amplification, Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

doi: 10.1038/s41598-019-38809-y

Figure Lengend Snippet: Expression of Il10 in EL4 cells transfected with siRNA for Ctse and CD4+ T cells isolated from B6 and MRL mice. ( A ) The knockdown and mRNA expression of Ctse in EL4 cells transfected with 5 μM control siRNA (si-Control) or Ctse siRNA (si-CTSE). *p < 0.05. ( B ) mRNA expression of Il10 in EL4 cells transfected with siRNAs for 24 hours and subsequently stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μM Ionomycin for 6 hours. ( C ) The concentration of IL-10 in supernatants and cell viability of cultured EL4 cells. EL4 cells were transfected with indicated siRNAs for 24 hours, stimulated with 10 ng/ml PMA and 1 μM Ionomycin for 12 or 24 hours. In cell viability, percentage of Trypan Blue negative cells is shown. *p < 0.05, ***p < 0.001. ( D ) mRNA expression of Il10 and Pdcd4 in CD4+ T cells isolated from B6 and MRL mice. ***p < 0.001.

Article Snippet: CD4+ T lymphocytes were purified using the RosetteSep Human CD4+ TCD4+ T Cell Enrichment Cocktail (Stem Cell Technologies).

Techniques: Expressing, Transfection, Isolation, Knockdown, Control, Concentration Assay, Cell Culture

Journal: Scientific Reports

Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

doi: 10.1038/s41598-019-38809-y

Figure Lengend Snippet: The increased levels of CTSE and IL10 transcripts in CD4+ T cells from SLE patients. ( A ) and ( B ) mRNA expression of CTSE and IL10 in CD4+ T cells isolated from healthy (n = 8) and the patients with SLE (n = 15). ( C ) mRNA expression of PDCD4 . There is no significant difference in healthy subjects and patients with SLE. ( D ) The simple correlation between the CTSE and IL10 transcript levels. *p < 0.05.

Article Snippet: CD4+ T lymphocytes were purified using the RosetteSep Human CD4+ TCD4+ T Cell Enrichment Cocktail (Stem Cell Technologies).

Techniques: Expressing, Isolation