Production of H₂S is downregulated in replicatively senescent cells.

<p>(<b>A</b>) Representative images of SA-β-Gal staining in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Scale bars, 100 μm. (<b>B</b>) Real-time PCR analysis of expression of <i>hTERT</i> in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>hTERT</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) NAD/NADH ratio in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Real-time PCR analysis of expression of <i>CBS</i> (<b>D</b>), <i>MST</i> (<b>E</b>), and <i>CSE</i> (<b>F</b>) in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>CBS</i>, <i>MST</i>, and <i>CSE</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>G</b>) 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. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p

NaHS-treatment increases expression of <i>NAMPT</i> and <i>SIRT1</i>.

<p>(<b>A</b> and <b>B</b>) The expression of <i>NAMPT</i> and <i>SIRT1</i> in young (PD: 5.9) and senescent (PD: 18.8) was assessed by real-time PCR and normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) 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. (<b>D</b> and <b>E</b>) 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 <i>NAMPT</i> and <i>SIRT1</i>. The expression levels of <i>NAMPT</i> and <i>SIRT1</i> were normalized to the levels of expression of <i>β-ACTIN</i>. (<b>F</b>) Immunoblotting of Nampt and Sirt1 in NaHS-treated young (PD: 5.9) aHDF cells. β-Actin was used as a loading control. (<b>G</b>) 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.</p

Exogenous H₂S increases PD and suppresses SA-β-Gal expression.

<p>(<b>A</b>) PD of cells treated without or with 1 μM NaHS. The population doubling of the first confluent cultures was designated as 0. (<b>B</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.</p

H₂S induces <i>hTERT</i> expression in a NAMPT/SIRT1-dependent manner.

<p>(<b>A</b> and <b>B</b>) Downregulation of <i>SIRT1</i> suppresses the expression of <i>hTERT</i>. Young (PD: 5.9) aHDF cells (3 x 10⁵ cells) were transfected with <i>SIRT1</i> 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 <i>SIRT1</i> (<b>A</b>) and <i>hTERT</i> (<b>B</b>). Data were normalized to the level of expression of <i>β-ACTIN</i>. The expression level of <i>SIRT1</i> and <i>hTERT</i> in cells treated with Scrambled siRNA without NaHS treatment was regarded as 1.0. (<b>C</b>) Downregulation of <i>NAMPT</i> suppresses the activity of SIRT1. Young (PD: 5.9) aHDF cells (3 x 10⁵ cells) were transfected with <i>NAMPT</i> 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. (<b>D</b>) Mode of action of H₂S in opposing senescence. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p

Exogenous H₂S increases the expression of <i>hTERT</i> as well as the activity of telomerase.

<p>(<b>A</b>) Real-time PCR analysis of the expression of <i>hTERT</i> in young (PD: 5.9) aHDF cells, treated with NaHS for 3 days. The expression of <i>hTERT</i> was normalized to the level of expression of <i>β-ACTIN</i>. Expression of untreated control was regarded as 1.0. (<b>B</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. (<b>C</b>) 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.).</p

Damage-Recovered (DR) cells show increase in H₂S and proliferation rate and exhibit tolerance to damage.

<p>(A) A scheme for isolation of Damage-Recovered (DR) cells. (B) Bax expression in H₂O₂ treated Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (C) Amount of H₂S released by DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.0005 in ANOVA statistical analysis. (D) H₂S staining of Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells with 5 µM H₂S fluorescent probe, HSN2. Scale bars, 50 µm. (E) PCR analysis of <i>CBS</i>, <i>CTH</i> and <i>MTS</i> genes in Pc, DR^{H2O2 W1} and DR^{H2O2 W3} HepG2 cells. (F) Western blot analysis of CBS and CTH in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (G) Western blot analysis of CBS in Pc and DR^{H2O2 W1}, DR^{H W1} and DR^{G W1} HepG2 cells. (H) Proliferation of HepG2 recovered from H₂O₂, DR^{H2O2 W1}, DR^{H2O2 W2} cells as a percentage of that in Pc cells. (I) Viability of Pc, DR^{H2O2 W1} and DR^{H2O2 W2} HepG2 cells with and without treatment with bleomycin. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S levels and bioenergetic changes in DR cells isolated from tumors.

<p>(A) H₂S levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (B) Expression of CBS and CTH in T^V and T^DR cells. (C) NAD⁺ levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (D) Nampt levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (E) ECAR in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the ECAR level in Pc. (F) ATP level in viable tumor cells (T^V) isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the ATP level in Pc. (G) Proliferation of T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the proliferation of Pc. Cells were seeded at a concentration of 2×10⁴ and the total number of cells was assessed after 24 and 48 hr of culture. (H) A scheme for H₂S-Nampt dependent bioenergetic circuit. Cell damage leads to increased H₂S and Nampt that coordinately lead to metabolic changes. These cells exhibit exponential growth and tolerance to damage. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

Endogenous hydrogen sulfide increases in response to acute damage in cancer cells.

<p>(A) Amount of H₂S released by 293 cells, fibroblasts (Fibro.), HepG2, MDA-MB-231 and MDA-MB-435S cells. (B) The levels of H₂S in HepG2, MDA-MB-231 and MDA-MB-435S Pc cells subjected to hypoxia (0.5% O₂, 18 hr), glucose deprivation (glucose free medium, 18 hr) or treatment with bleomycin (35 nM, 18 hr), H₂O₂ (800 µM, 3 hr). Data are expressed as percent of H₂S released from untreated cells. (C) Intracellular CBS and Bax (left panel) assessed by western blot analysis in MDA-MB-435S Pc cells, and Pc cells treated with 400 or 800 µM of H₂O₂ for 3 hr. (D) The level of CBS and γH2AX with or without treatment with H₂O₂ (800 µM, 3 hr) in MDA-MB-435S cells. Protein density was normalized using β-Actin. (E) Amount of H₂S and cell viability after treatment with a range of concentration of H₂O₂. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S increases ECAR, ATP, NAD⁺ and Nampt in a dose-dependent manner in cancer cells.

<p>(A) ECAR in Pc HepG2 cells treated for 48 hr with 0, 1, and 100 µM of NaHS and DR^{H W1} HepG2 cells. (B) Comparison of ATP levels in Pc HepG2 cells treated for 48 hr with 0, 10 and 100 µM of NaHS, DR^{H2O2 W1} HepG2 cells and Pc MDA-MB-231 cells treated for 48 hr with 0, 10 and 100 µM of NaHS. Data are expressed as a percent of level of ATP in untreated cells. (C) Levels of NAD⁺ in HepG2 and MDA-MB-231 Pc cells treated with 0, 10, and 100 µM of NaHS for 48 hr. (D) Western blot analysis of intracellular Nampt in MDA-MB-231 Pc cells treated for 48 hr with 0 and 100 µM of NaHS. (E) qPCR analysis of <i>NAMPT</i> expression in Pc HepG2 cells treated for 48 hr with 0 and 100 µM of NaHS. (F) Quantitation of intracellular Nampt by ELISA in Pc HepG2 cells treated for 48 hr with 0, 10 and 100 µM of NaHS. (G) Viability in HepG2 cells pre-treated for 24 hr with 0, 1, and 10 mM of NaHS and then subjected to H₂O₂ (800 µM) or bleomycin (35 nM, 18 hr). Viability was assessed by Trypan blue exclusion. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S-Nampt pathway regulates bioenergetics.

<p>(A) Amount of H₂S released from DR^{H W1} HepG2 cells treated with CTH inhibitor, PAG (100 µM, 18 hr), and Nampt inhibitor, FK866 (200 nM, 24 hr). (B) Western blot analysis of CBS in DR cells in the absence and presence of FK866 (200 nM, 24 hr). (C) Western blot analysis of CTH in DR cells in the absence and presence of FK866 (200 nM, 24 hr). (D) ATP levels in DR^{H2O2 W3} HepG2 cells in the absence (−) and presence (+) of PAG (100 µM, 18 hr). (E) ATP levels in Pc, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells in the absence and presence of FK866 (200 nM, 24 hr). (F) Western blot analysis of Nampt in DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells treated with CBS inhibitor, CHH (500 µM, 18 hr) or CTH inhibitor, PAG (100 µM, 18 hr). *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p