Journal: Aging (Albany NY)

Article Title: GRSF1 suppresses cell senescence

doi: 10.18632/aging.101516

Figure Lengend Snippet: GRSF1 knockout or silencing elevates DNA damage, reduces DNA replication, and delays cell cycle progression. ( A ) Mitochondrial superoxide was assessed by fluorescent microscopy in shRNA-transfected WI-38 fibroblasts expressing normal (shCTRL) or reduced (shGRSF1) levels of GRSF1. The mitochondrial superoxide probe MitoSOX Red was employed to measure the relative production of superoxide, which was quantified as the average (avg.) fluorescence intensity ( left ); representative fluorescence images of the oxidized MitoSOX fluorescence signal (red) are shown ( right ). GRSF1 levels in infected WI-38 cells were assessed by Western blot analysis ( left ). ( B ) DNA double-strand breaks (DSBs) were monitored by measuring the phosphorylated histone H2A.X on serine 139 (γH2A.X) by using flow cytometry-based analysis at 4 and 8 days (D4 and D8) after IR (10 Gy). ( C ) WI-38 fibroblasts were infected with lentiviruses expressing shGRSF1 or shGRSF1; following ionizing irradiation (10 Gy) DNA damage was assessed by immunofluorescence to visualize 53BP1 foci ( right ); foci were counted in WI-38 fibroblasts expressing shCTRL or shGRSF1 that had been left untreated (-IR) or had been exposed to IR (+IR) ( Left ). ( D ) WI-38 fibroblasts that expressed constitutively shGRSF1 or shCTRL were synchronized at G1/S phase by double-thymidine block, released, and collected for analysis of cell cycle progression at the indicated time points by flow cytometry; the progression of cells through the division cycle was quantified (G2/M, G1/S). ( E ) WI-38 fibroblasts were infected with shGRSF1 or shCTRL lentiviruses and expanded for three weeks; the incorporation of [ 3 H]-thymidine was then quantified as a measure of cell proliferation. Data in ( B , C , E ) represent the means and S.D. from three independent experiments.

Article Snippet: To generate THP-1 cells expressing constitutively lower levels of GRSF1, cells were transduced with lentiviral particles expressing shGRSF1 or shCTRL (Origene) one week later they were treated with puromycin (0.5 μg/mL) to select for cells stably expressing integrated vectors.

Techniques: Knock-Out, Microscopy, shRNA, Transfection, Expressing, Fluorescence, Infection, Western Blot, Flow Cytometry, Irradiation, Immunofluorescence, Blocking Assay

Journal: Aging (Albany NY)

Article Title: GRSF1 suppresses cell senescence

doi: 10.18632/aging.101516

Figure Lengend Snippet: Silencing GRSF1 promotes senescence and IL6 production. ( A ) Three weeks after silencing GRSF1 by infection with shGRSF1- or shCTRL-expressing lentiviruses, SA-β-gal activity was assessed by light microscopy ( left ) and secreted IL6 was quantified by ELISA ( right ). (B) Pre-senescent WI-38 fibroblasts were transfected with siGRSF1 or siCTRL; SA-β-gal activity (micrographs) was assessed at day 8 (D8) and D15 after transfection. At D8, GRSF1 and GAPDH levels in WCL were assessed by Western blot analysis ( right ). (C,D) WI-38 cells transfected with siGRSF1 or siCTRL were irradiated (IR, 10 Gy) and subsequently cultured for 10 days; whereupon SA-β-gal activity was assessed ( C ); proteins in WCL prepared at D5 and D18 were assessed by Western blot analysis ( D , left ). RNA was extracted at D7 and D20, and IL6 mRNA levels were quantified by RT-qPCR analysis ( D , right ). ( E ) After transfection with siGRSF1 or siCTRL and irradiation, WI-38 cells were cultured in normal medium for 14 days and in serum-free condition for 24 h ( left ). CM was then collected, and the secreted IL6 was assayed by AlphaLISA ( right ). Data in ( D , E ) represent the means ±S.D. from three independent experiments.

Article Snippet: To generate THP-1 cells expressing constitutively lower levels of GRSF1, cells were transduced with lentiviral particles expressing shGRSF1 or shCTRL (Origene) one week later they were treated with puromycin (0.5 μg/mL) to select for cells stably expressing integrated vectors.

Techniques: Infection, Expressing, Activity Assay, Light Microscopy, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Irradiation, Cell Culture, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: GRSF1 suppresses cell senescence

doi: 10.18632/aging.101516

Figure Lengend Snippet: Loss of GRSF1 increases the paracrine actions of IL6. ( A ) THP-1 monocytes expressing lentiviral shGRSF1 or shCTRL were maintained in normal culture medium (containing 10% FBS), and the amount of IL6 secreted into the CM was measured by ELISA. ( B ) Human monocyte THP-1 cells were infected with shGRSF1 or shCTRL lentiviruses to produce different steady-state levels of GRSF1 constitutively. The levels of IL6 secretion by these cells was measured by ELISA; treatments with LPS (50 ng/mL) for 24, 48, and 72 h were included as positive controls for IL6 production. ( C - E ) Schematic of the co-culture setup. Proliferating (PDL20-25) or senescent (PDL55-60) WI-38 fibroblasts were plated in the lower chamber, and THP-1 cells constitutively expressing different levels of GRSF1, as explained in ( A ), were plated in the upper chamber. The chambers were separated by a selectively permeable membrane with 0.4 µm-diameter pores ( C ). Lentiviral shRNA-expressing THP-1 cells were co-cultured with either proliferating or senescent WI-38 fibroblasts for 48 h, and the levels of IL6 mRNA in THP-1 cells ( D ) and WI-38 cells ( E ) were quantified by RT-qPCR analysis. Data in A , D , E represent the means ±S.D. from three independent experiments. ( F ) Model: we propose that loss of GRSF1 contributes to cellular senescence characterized by oxidative stress, DNA damage, growth suppression, and IL6 production.

Article Snippet: To generate THP-1 cells expressing constitutively lower levels of GRSF1, cells were transduced with lentiviral particles expressing shGRSF1 or shCTRL (Origene) one week later they were treated with puromycin (0.5 μg/mL) to select for cells stably expressing integrated vectors.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Co-Culture Assay, Membrane, shRNA, Cell Culture, Quantitative RT-PCR