Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts

doi: 10.1152/ajplung.00473.2016

Figure Lengend Snippet: Effect of SIRT7 silencing on collagen and α-SMA protein levels. A: normal adult lung fibroblasts from three healthy control donors (C1–C3) were transfected with scrambled control or SIRT7 siRNAs, as indicated, and Western blotting for collagen type I protein (COL1) performed after 48 or 72 h. B: α-SMA protein levels for two healthy control donors (C1, C2) 72 h after silencing with scrambled or SIRT7 siRNAs. Noncontiguous gels are demarcated by white spaces. C: immunofluorescent staining for α-SMA in fibroblasts from a healthy control donor (different from the two controls shown in B) 48 h after transfection with scrambled or SIRT7 siRNAs. Note the increases in collagen and α-SMA in SIRT7-silenced fibroblast cultures.

Article Snippet: Western blots were performed with primary rabbit antibodies to SIRTs 1–3 and 5–7, phosphorylated and total Smad2/3, GAPDH, β-actin, and MEK1/2 from Cell Signaling Technology (Danvers, MA), HDAC2 and α-SMA from Abcam (Cambridge, MA), and collagen type I from Rockland Antibodies and Assays (Limerick, PA).

Techniques: Transfection, Western Blot, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts

doi: 10.1152/ajplung.00473.2016

Figure Lengend Snippet: Effect of SIRT7 overexpression on the levels of fibrosis-associated molecules. A: RT-qPCR for SIRT7, COL1A1, COL1A2, and COL3A1 mRNA levels in cultured primary pulmonary fibroblasts from a healthy control at indicated times after stimulation with rhTGF-β1. Before stimulation, cells were transfected with equal amounts of a SIRT7-encoding or control noncoding (NULL) plasmid; both plasmids had a similar backbone. Cells were stimulated with TGF-β 48 h after transfection. Means ± SD of duplicate measurements are shown; the experiment was repeated in fibroblast cultures from two different donors on two separate occasions. The mRNA levels of each target were normalized to the levels of 18S rRNA and further normalized to the NULL sample without TGF-β at each time point. Significant differences (P < 0.05) from NULL-transfected cells without TGF-β stimulation are indicated by asterisks, and significant differences from NULL-transfected cells with TGF-β are indicated by daggers. B: RT-qPCR for α-SMA and connective tissue growth factor (CTGF) in cultured primary pulmonary fibroblasts from two healthy controls (C1, C2) transfected with NULL or SIRT7 plasmids and stimulated with rhTGF-β1 48 h later. Analyses were performed 24 h after TGF-β stimulation. Significant differences (P < 0.05) are indicated. C: changes in collagen type I protein levels in cultured primary pulmonary fibroblasts from a healthy control (C) and patient with IPF (P) following overexpression of SIRT7. Fibroblasts were electroporated with a control noncoding (NULL) or SIRT7-encoding plasmid and Western blots for SIRT7, COL1, and GAPDH performed at the indicated times. Note the decreases in collagen levels in SIRT7-overexpressing cultures. These experiments were performed in primary cultures from four different healthy donors on at least four separate occasions and two donors with IPF with similar results. Noncontiguous gels are demarcated by white spaces. D: α-SMA protein levels in two healthy controls (C1 and C2) and a patient with IPF (P) at indicated times after transfection with NULL or SIRT7-encoding plasmids. Note the decreases in α-SMA in cultures overexpressing SIRT7. E: immunofluorescent staining for α-SMA in normal lung fibroblasts transfected with NULL or SIRT7 plasmids and stimulated with rhTGF-β 48 h later. Cells were fixed, and staining was performed 48 h after TGF-β stimulation. SIRT7 overexpression appears to slightly decrease α-SMA expression both with and without TGF-β stimulation. F: effects of SIRT7 overexpression combined with rhTGF-β1 stimulation on COL1 and α-SMA protein levels in healthy control lung fibroblasts. Cells were electroporated with NULL- or SIRT7-encoding plasmids and, after 48 h, stimulated with rhTGF-β or cultured with no stimulation (medium) for an additional 72 h. Note that the stimulating effect of TGF-β on collagen and α-SMA is suppressed by SIRT7. F, bottom: densities for COL1 and α-SMA normalized to GAPDH are shown.

Article Snippet: Western blots were performed with primary rabbit antibodies to SIRTs 1–3 and 5–7, phosphorylated and total Smad2/3, GAPDH, β-actin, and MEK1/2 from Cell Signaling Technology (Danvers, MA), HDAC2 and α-SMA from Abcam (Cambridge, MA), and collagen type I from Rockland Antibodies and Assays (Limerick, PA).

Techniques: Over Expression, Quantitative RT-PCR, Cell Culture, Transfection, Plasmid Preparation, Western Blot, Staining, Expressing