Fig 1.
Production of H2S is downregulated in replicatively senescent cells.
(A) Representative images of SA-β-Gal staining in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Scale bars, 100 μm. (B) Real-time PCR analysis of expression of hTERT in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of hTERT was normalized to the expression level of β-ACTIN. (C) NAD/NADH ratio in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Real-time PCR analysis of expression of CBS (D), MST (E), and CSE (F) in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of CBS, MST, and CSE was normalized to the expression level of β-ACTIN. (G) 1 x 106 cells of young (PD: 5.9) and senescent (PD: 18.8) aHDF cells were incubated in PBS at 37°C for 1 hour and then H2S was measured in culture supernatants. Mean values are shown along with error bars. *; p<0.05, **; p<0.005, ***; p<0.0005, n.s.; not significant.
Fig 2.
Exogenous H2S increases the expression of hTERT as well as the activity of telomerase.
(A) Real-time PCR analysis of the expression of hTERT in young (PD: 5.9) aHDF cells, treated with NaHS for 3 days. The expression of hTERT was normalized to the level of expression of β-ACTIN. Expression of untreated control was regarded as 1.0. (B) Immunoblotting of hTERT in aHDF cells without or with 1 μM NaHS for 7 days. 100 μg of the indicated nuclear extracts were subjected for immunoblotting. β-Actin was used as a loading control. (C) Telomerase activity in young (PD: 3.2) aHDF cells without or with treated with 1 μM NaHS for 7 days. Positive control was MDA-MB-231 cell lysate, and negative control was buffer alone. Bottom panel shows quantified means ± error bars from three independent assays. Relative activity of telomerase was calculated by dividing the density of all ladders to the density of the bands in internal control, indicated as internal control (I.C.).
Fig 3.
Exogenous H2S increases PD and suppresses SA-β-Gal expression.
(A) PD of cells treated without or with 1 μM NaHS. The population doubling of the first confluent cultures was designated as 0. (B) Representative images of SA–β-Gal staining in cells shown in Fig 3A. Mean values ± error bars of number of SA-β-Gal positive cells are shown on the right-upper corner of each image. *; p<0.05, ***; p<0.0005, n.s.; not significant. Scale bars, 100 μm.
Fig 4.
NaHS-treatment increases expression of NAMPT and SIRT1.
(A and B) The expression of NAMPT and SIRT1 in young (PD: 5.9) and senescent (PD: 18.8) was assessed by real-time PCR and normalized to the expression level of β-ACTIN. (C) Immunoblotting of Nampt and Sirt1 in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. β-Actin was used as a loading control. (D and E) Young (PD: 5.9) aHDF cells were treated without and with NaHS for 3 days, and RNA samples were then subjected to real-time PCR for assessment of NAMPT and SIRT1. The expression levels of NAMPT and SIRT1 were normalized to the levels of expression of β-ACTIN. (F) Immunoblotting of Nampt and Sirt1 in NaHS-treated young (PD: 5.9) aHDF cells. β-Actin was used as a loading control. (G) NAD/NADH ratio in young (PD: 5.9) aHDF cells treated without and with NaHS for 7 days. Data were normalized to the total amount of protein.
Fig 5.
H2S induces hTERT expression in a NAMPT/SIRT1-dependent manner.
(A and B) Downregulation of SIRT1 suppresses the expression of hTERT. Young (PD: 5.9) aHDF cells (3 x 105 cells) were transfected with SIRT1 siRNA for 2 days, and these were treated without or with NaHS for 3 days. Total RNAs from these cells were subjected to real-time PCR analysis for SIRT1 (A) and hTERT (B). Data were normalized to the level of expression of β-ACTIN. The expression level of SIRT1 and hTERT in cells treated with Scrambled siRNA without NaHS treatment was regarded as 1.0. (C) Downregulation of NAMPT suppresses the activity of SIRT1. Young (PD: 5.9) aHDF cells (3 x 105 cells) were transfected with NAMPT siRNA for 2 days, and then the cells were treated without or with 1 μM NaHS for 3 days. Nuclear proteins were extracted and used for measurement of SIRT1 activity. Mean values ± error bars were normalized to the amount of total cell protein. (D) Mode of action of H2S in opposing senescence. *; p<0.05, **; p<0.005, ***; p<0.0005, n.s.; not significant.