Journal: eLife

Article Title: The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack

doi: 10.7554/eLife.41301

Figure Lengend Snippet:

Article Snippet: Cell line ( Rattus norvegicus ) , Normal rat kidney (NRK) fibroblast cell , ATCC , ATCC: CRL-1570; RRID: CVCL_2144 , .

Techniques: Recombinant, Plasmid Preparation, Antibody Labeling, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

doi: 10.1111/j.1582-4934.2012.01610.x

Figure Lengend Snippet: Effects of conditioned MSCs on expression of α-SMA and fibronectin in renal interstitial fibroblast NRK-49F. NRK-49F cells were cultured without or with 15 ng/ml TGF-β1 alone or co-culture with conditioned MSCs and/or ascorbic acid 2- phosphate for 3 days. ( A ) Representative Western blot analysis and relative bar graph analysis for α-SMA and β-tubulin level. ( B ) Representative Western blot and relative bar graph analysis of fibronectin protein level in NRK-49F after various treatments. * P < 0.05 versus normal control; ** P < 0.05 versus TGF-β1 treated.

Article Snippet: Rat renal proximal tubular cells (NRK-52E) and normal rat kidney interstitial fibroblast cells (NRK-49F) were purchased from the Bioresource Collection and Research Center of the Food Industry Research Institute, Taiwan.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Western Blot, Control

Journal: Research

Article Title: Epigenetic Suppression of RASAL1 by HDAC3 and Cofactor YY1 Promotes Fibroblast–Myofibroblast Transition and Renal Fibrosis

doi: 10.34133/research.1073

Figure Lengend Snippet: Fibrotic kidneys induced by UUO or AAI display aberrant up-regulation of HDAC3 and suppression of RASAL1 in renal fibroblasts. C57BL/6 mice were subjected to UUO for 7 d or AAI (5 mg/kg, intraperitoneal injection every other day) for 14 d (6 mice in each group). (A) Representative photomicrographs of kidney sections stained with Masson’s trichrome and Sirius Red from sham-, UUO-, control-, and AAI-treated mice. The arrows indicate collagen-stained fibrotic areas. (B) Quantitation of renal fibrosis in (A). (C) Western blots of renal tissues for Colla 1, α-SMA, HDAC3, and RASAL1. Two representative samples from each group were shown. β-Actin served as loading control. (D) Quantifications of (C). (E) RT-PCR of renal tissues from sham-, UUO-, control-, and AAI-treated mice ( n = 6) for Rasal1 mRNAs. Gapdh served as the internal control. Two random samples from each group were shown. The right panel was the quantification. bp, base pairs. Data were presented as means ± SEM based on 6 renal samples. * P = 0.05, 2-tailed unpaired t test for (B), (D), and (E). (F) Representative photomicrographs of kidney sections from sham-, UUO-, control-, and AAI-treated mice stained for RASAL1 by IHC staining. Positive staining was indicated by arrows. (G) Western blotting. NRK-49F and mouse primary renal fibroblast (PRF) cells were treated with AAI (30 μM) for 24 h, and then the cell lysates were tested for HDAC3 and RASAL1. The quantitation was on the right side. Data were presented as means ± SD of 6 repeated cell assays. *P < 0.05, 2-tailed unpaired t test. (H) Representative photomicrographs of kidney sections from renal patients (mild injury and CKD) stained by Masson’s trichrome for renal fibrosis and IHC for HDAC3 and RASAL1. The arrows indicate collagen-stained fibrotic areas and positively stained fibroblast-rich areas. (I) Quantifications of (H) presented as means ± SEM of 6 mild injury and 10 CKD samples. Data were presented as means ± SEM based on 6 renal samples. * P < 0.05, 2-tailed unpaired t test.

Article Snippet: Human embryonic kidney (HEK) 293T cells (American Type Culture Collection, USA) were cultured in DMEM, and rat renal fibroblast NRK-49F cells (Procell Life Science & Technology, Wuhan, China) and mouse primary renal fibroblasts were cultured in DMEM/F12 medium.

Techniques: Injection, Staining, Control, Quantitation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: Research

Article Title: Epigenetic Suppression of RASAL1 by HDAC3 and Cofactor YY1 Promotes Fibroblast–Myofibroblast Transition and Renal Fibrosis

doi: 10.34133/research.1073

Figure Lengend Snippet: HDAC3 inhibits RASAL1 transcription in renal fibroblasts. (A) NRK-49F cells were transfected with either a control plasmid (vector) or a plasmid overexpressing Flag-tagged HDAC3 (F-HD3). Twenty-four hours later, the cell lysates were assayed for HDAC3, RASAL1, and Colla 1. β-Actin served as the internal control. (B) NRK-49F cells were treated with TGFβ (10 ng/ml) in the presence or absence of RG (10 μM) for 24 h, and then the cell lysates were tested for HDAC3, RASAL1, and Colla 1. (C) Quantitation of Western blots in (B). (D) Western blotting. NRK-49F cells transfected with vector or Flag-tagged HDAC3 plasmids for 24 h, followed by treatment with TGFβ (10 ng/ml) and/or RG (10 μM) for another 24 h. Cell lysates were assayed with antibody against HDAC3, RASAL1, and α-SMA. (E) Quantitation of (D). (F) NRK-49F cells were treated with TGFβ (10 ng/ml) in presence or absence of RG (10 μM) for 24 h, and the mRNA of Rasal1 was analyzed by RT-PCR. Gapdh served as the internal control. The bottom panel is the quantitation. (G) Luciferase assay. HEK293T cells were transfected with either a positive control plasmid 3TP-luc or the Rasal1 promoter reporter Rasal1 p-luc together with a Renilla luciferase reporter and then treated with TGFβ (10 ng/ml) with or without RG (10 μM) for 24 h. Cell lysates were assayed for luciferase activities. (H) Schematic representation of the mouse Rasal1 promoter region spanning −300 to +243 relative to the transcriptional start site. The CpG island (gray-shaded area) and positions of qMSP primers (boxed regions) are depicted. (I) qMSP analysis of the Rasal1 promoter methylation in renal tissues from sham and UUO mice (7 d) (left) and in NRK-49F renal fibroblasts treated with TGFβ (10 ng/ml; 24 h) (right) with or without RG (10 mg/kg in mice; 10 μM in cell culture). (J) Representative agarose gel analysis of methylated (Met), unmethylated (unMet) and input PCR products for mouse tissue and NRK-49F cells mentioned above. (K) Quantitation of (J). Data were presented as means ± SD of 3 repeated cell assays or means ± SEM based on 6 renal samples. * P < 0.05, 2-tailed unpaired t test (3TP-luc) or 2-way ANOVA ( Rasal1 p-luc) (C, E, F, I, and K).

Article Snippet: Human embryonic kidney (HEK) 293T cells (American Type Culture Collection, USA) were cultured in DMEM, and rat renal fibroblast NRK-49F cells (Procell Life Science & Technology, Wuhan, China) and mouse primary renal fibroblasts were cultured in DMEM/F12 medium.

Techniques: Transfection, Control, Plasmid Preparation, Quantitation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Luciferase, Positive Control, Methylation, Cell Culture, Agarose Gel Electrophoresis

Journal: Research

Article Title: Epigenetic Suppression of RASAL1 by HDAC3 and Cofactor YY1 Promotes Fibroblast–Myofibroblast Transition and Renal Fibrosis

doi: 10.34133/research.1073

Figure Lengend Snippet: HDAC3 inhibition of Rasal1 transcription involves YY1. (A) Western blots of renal tissues from sham-, UUO (7 d)-, control-, and AAI (14 d)-treated mice ( n = 6) for YY1. The quantitation was on the right side. (B) Molecular docking between HDAC3 and YY1. The binding energy was analyzed by MM-GBSA. (C) Co-IP assay. Renal tissue homogenates from sham-, RG-, UUO-, or RG/UUO mice ( n = 6) were assayed for HDAC3 and YY1 as input controls (the bottom 3 blots). Subsequently, the same tissue lysates were immunoprecipitated with isoform-matched immunoglobulin (Ig) or antibodies (IPab) specific to HDAC3 (HD3) or YY1, and then immunoprecipitants were reciprocally assessed for HDAC3 and YY1 by Western blotting (the top 2 blots). (D) Quantification of Co-IP in (C). Data were presented as means ± SEM based on 3 renal samples from each group. (E) NRK-49F cells were transfected with control lentivirus (siC) and 3 YY1 knockdown lentiviruses (si1, si2, and si3) for 48 h. Western blotting was performed on cell lysates to measure YY1 protein levels. The bottom panel was the quantification of protein. (F) NRK-49F cells were transfected with control lentivirus (siC) and YY1 knockdown lentivirus (siR-Yy1, si3) for 24 h, followed by treatment with TGFβ (10 ng/ml) for additional 24 h. Cell lysates were assayed for RASAL1 and YY1 expression by Western blotting. (G) The top panel shows the YY1 binding motif sequence logo from JASPAR, while the bottom panel depicts a schematic diagram of YY1 binding to the Rasal1 promoter. TSS, transcription start site. (H) ChIP assay. The control, UUO, or AAI mice were treated with or without RG (10 mg/kg). The renal tissues indicated above were immunoprecipitated with antibodies to HDAC3, YY1 and Ac-H3, and then the precipitated genomic DNA (input) and the antibody-bound DNAs were PCR amplified with primers covering the YY1 motif on Rasal1 promoter. The RT-PCR products were analyzed on an agarose gel (left). RT-qPCR (right) was normalized to input DNA and presented as fold changes relative to sham. (I) Luciferase assay. HEK293T cells were transfected with the murine Rasal1 promoter reporter ( Rasal1 p-luc) or the m Rasal1 p-luc plus a Renilla luciferase plasmid. After 24 h of YY1 knockdown and control lentivirus treatment, cells were treated with TGFβ (10 ng/ml) for additional 24 h, and the luciferase activities were measured and normalized to Renilla luciferase activities. Data were presented as means ± SEM based on 6 renal samples or means ± SD of 3 repeated cell assays. * P < 0.05, one-way ANOVA (A and E), 2-way ANOVA (D, F, and H), or 3-way ANOVA (I).

Article Snippet: Human embryonic kidney (HEK) 293T cells (American Type Culture Collection, USA) were cultured in DMEM, and rat renal fibroblast NRK-49F cells (Procell Life Science & Technology, Wuhan, China) and mouse primary renal fibroblasts were cultured in DMEM/F12 medium.

Techniques: Inhibition, Western Blot, Control, Quantitation Assay, Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Transfection, Knockdown, Expressing, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Luciferase, Plasmid Preparation

Journal: Research

Article Title: Epigenetic Suppression of RASAL1 by HDAC3 and Cofactor YY1 Promotes Fibroblast–Myofibroblast Transition and Renal Fibrosis

doi: 10.34133/research.1073

Figure Lengend Snippet: RASAL1 derepression is essential for the antifibrosis function of HDAC3 inhibition in vitro and in vivo . (A) Top: NRK-49F cells were treated with siC, si1, or si2 for 48 h. Western blotting of the cell lysates was assayed for RASAL1 proteins. Bottom: C57BL/6 mice were treated with Ch-siC, Ch-si1, or Ch-si2 once by intravenous injection, 6 mice in each group. Seven days later, Western blotting was performed on mouse renal tissues to detect RASAL1 proteins. (B) Western blotting. NRK-49F cells were infected with siC or si2 for 48 h, and then the cells were treated with TGFβ (10 ng/ml) and/or RG (10 μM) for 24 h. Cell lysates were assayed with antibody against HDAC3, RASAL1, α-SMA, and Colla 1. (C) Quantifications of Western blots in (B). Data were presented as means ± SD of 3 repeated cell assays. * P < 0.05, 3-way ANOVA followed by Tukey’s post hoc test. (D) Mice receiving Ch-siC or Ch-si2 were subgrouped into sham, RG, UUO, or RG/UUO ( n = 6). Representative photomicrographs of kidney sections were stained by Sirius Red. The black arrows indicate fibrotic areas. (E) Quantification of (D). Data were presented as scatter plot. *P < 0.05, 3-way ANOVA followed by Tukey’s post hoc test. (F) Western blotting. The renal tissues were assayed for HDAC3, RASAL1, α-SMA, and Colla 1. Two samples from each group were shown. (G) Quantifications of (F). Data were presented as means ± SEM based on 6 renal samples. *P < 0.05, 2-way ANOVA. (H) A schematic diagram of sequential HDAC3 elevation, RASAL1 suppression, and FMT during renal fibrosis. Elevated HDAC3, in association with YY1, induces hypoacetylation and transcriptional suppression of the Rasal1 promoter, resulting in persistent FMT and development of renal fibrosis (red lines). Conversely, selective HDAC3 inhibition (HDAC3i) by RG mitigates Rasal1 suppression (black lines), FMT, and renal fibrosis.

Article Snippet: Human embryonic kidney (HEK) 293T cells (American Type Culture Collection, USA) were cultured in DMEM, and rat renal fibroblast NRK-49F cells (Procell Life Science & Technology, Wuhan, China) and mouse primary renal fibroblasts were cultured in DMEM/F12 medium.

Techniques: Inhibition, In Vitro, In Vivo, Western Blot, Injection, Infection, Staining