Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: C57BL/6 mice were injected with cisplatin (cis3d) or saline (control) to collect kidney cortex and outer medulla 3 days later for the isolation of genomic DNAs, which were subjected to genome-wide DNA methylation analysis via reduced representation bisulfite sequencing (RRBS). 200 bp non-overlapping windows were used to identify differentially methylated regions (DMRs). DMRs were identified with a methylation difference over 0.25. (A) Representative heat map. Red color indicates high levels of DNA methylation and blue color indicates low levels of methylation. (B) The number of DMRs identified in cisplatin-induced AKI. (C) Genome-wide distribution of the DMRs. The results were from 2 separate experiments with 2 pairs of littermate mice.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Injection, Isolation, Genome Wide, DNA Methylation Assay, Methylation Sequencing, Methylation

Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: RPTC cells were treated with 20 μM cisplatin in the absence or presence of 1 μM 5-aza pretreatment. (A) Immunoblot. After 16h cisplatin treatment, whole cell lysate was collected for immunoblot analysis of indicated proteins. Cyclophilin B was used as protein loading control. (B) Cell morphology. After 16h cisplatin treatment, cells were stained with Hoechst 33342 to record cellular and nuclear morphology by phase contrast and fluorescence microscopy, respectively. Scale bar: 200 μm. (C) Percentage of cell apoptosis. The cells with typical apoptotic morphology were counted to determine the % of apoptosis. Data in (C) are presented as mean ± SD; n = 4. *P < 0.05, significantly different from control; #P < 0.05, significantly different from the cisplatin-only group.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot, Staining, Fluorescence, Microscopy

Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: PT-DNMT1-WT and PT-DNMT1-KO littermate mice were injected with 30 mg/kg cisplatin or saline as control. Kidney tissues and blood samples were collected at day 4 after cisplatin injection for analysis. (A) Serum creatinine. (B) Representative H&E images of kidney cortex from PT-DNMT1-WT and -KO mice. Scale bar: 50 μm. (C) Kidney tissue damage score in cisplatin- treated PT -DNMT1-WT and -KO mice. (D) Representative images of cleaved caspase 3 immunohistochmical staining. Scale bar: 50 μm. (E) Quantitative analysis of cleaved caspase 3 immunohistochemical staining. Quantitative data in (A) and (C) are expressed as mean ± SD (n=10). *P < 0.05, significantly different from PT-DNMT1-WT mice.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Injection, Staining, Immunohistochemical staining

Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: Primary proximal tubular cells were isolated from PT-DNMT1-WT or -KO mice. Then primary cells were treated with 50 μM cisplatin for 24 hours. (A) Cell morphology. After 24h cisplatin treatment, cells were stained with Hoechst 33342 to record cellular and nuclear morphology by phase contrast and fluorescence microscopy, respectively. (B) Percentage of apoptosis. The cells with typical apoptotic morphology were counted to determine the % of apoptosis. Scale bar: 200 μm. (C) Immunoblot analysis of Caspase 3 activation. After 24h cisplatin treatment, whole cell lysate was collected for immunoblot analysis of DNMT1, Caspase 3, and Cyclophilin B as protein loading control. Caspase 3 activation is indicated by the appearance of cleaved (active) fragment of caspase 3. Data in (B) are presented as mean ± SD; n= 3. *P < 0.05, significantly different from control DNMT1-WT cells; #P < 0.05, significantly different from the cisplatin treated DNMT1-WT cells.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Isolation, Staining, Fluorescence, Microscopy, Western Blot, Activation Assay

Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: In (A), (B) and (C), male C57BL/6 mice were injected with 30 mg/kg cisplatin or saline as control. (A) The RRBS result of Irf8 gene methylation changes shown by UCSC genome browser screenshot. The hypomethylated CpG sites following cisplatin-treatment are highlighted in green. (B) Real-time PCR analysis to verify Irf8 induction in kidney cortical tissues following cisplatin treatment. (C) Immunoblot analysis of Irf8 expression in renal cortical lysate. Cyclophilin B was used as a protein loading control. Irf8 signals were then analyzed by densitometry (bottom). After normalization with cylcophilin B, the protein signal of lane 1 was arbitrarily set as 1, and the signals of other lanes were normalized with lane 1 to calculate fold changes. (D) RPTC cells were treated with 20 μM cisplatin for 16 hours in the absence or presence of 5-aza pretreatment. Irf8 expression was shown by real-time PCR (top) and immunoblot (bottom) analysis. (E) PT-DNMT1-WT or -KO littermate mice were injected with 30 mg/kg cisplatin or saline as control. Irf8 mRNA level in the cortical and outer medulla total RNA was detected by real-time PCR. Data in (B) are expressed as median ± interquartile range; n=7. Data in (C) and (D) are expressed as mean ± SD; n=4. *P < 0.05, significantly different from control; #P < 0.05, significantly different from the cisplatin-only group. Data in (E) are presented as mean ± SD; n=5. *P<0.05, significantly different from control PT-DNMT1-WT mice; #P<0.05, significantly different from control PT-DNMT1-KO mice.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Injection, Methylation, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Kidney international

Article Title: DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes including interferon regulatory factor 8

doi: 10.1016/j.kint.2017.03.038

Figure Lengend Snippet: Irf8 shRNA and scrambled sequence plasmids were separately transfected into RPTC cells to select stably transfected cells. The cells were then treated with 20 μM cisplatin for 20 hours. (A) Immunoblot (top) and real-time PCR (bottom) analysis of Irf8 to confirm the silencing of Irf8. In immunoblot, cyclophilin B was used as protein loading control. (B) Cell morphology. Cells were stained with Hoechst 33342 to record cellular and nuclear morphology by phase contrast and fluorescence microscopy, respectively. Scale bar: 200 μm. (C) Percentage of cell apoptosis. The cells with typical apoptotic morphology were counted to determine the % of apoptosis. (D) Immunoblot analysis of Caspases 3 and Cyclophilin B as protein loading control. Data in (A) and (C) are presented as mean ± SD; n = 3. *P > 0.05, significantly different from control scrambled sequence-transfected cells. #P > 0.05, significantly different from cisplatin-treated scrambled sequence-transfected cells. $P>0.05, significantly different from control Irf8 shRNA sequence-transfected cells.

Article Snippet: Fluorescein-labeled lotus tetragonolobus lectin (LTL) was bought from Vector Labs (FL-1321). cis-Diammineplatinum(II) dichloride crystalline (Cisplatin, P4394-1G) and 5-aza-2′-deoxycytidine (A3656-50MG) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: shRNA, Sequencing, Transfection, Stable Transfection, Western Blot, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Microscopy