Cell cycle restoration after DNA damage in cancer cells.

<p>SCR or SLD5 siRNA transfected B16 (<b>A, B</b>) or Colon26 (<b>C, D</b>) cells were treated with DMSO (<b>A,</b><b>C</b>) or etoposide (<b>B,</b><b>D</b>) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110483#pone-0110483-g006" target="_blank">Figure 6</a> and the same number of living cells as indicated were cultured with fresh medium for 72 h. Cell numbers were recorded. Data represent the mean ± SD.*, <i>P</i><0.05 (n = 3).</p

Attenuation of SLD5 expression induces marked DNA damage in cancer cells.

<p>Western blot analysis of γ-H2AX expression in SCR or SLD5 siRNA-transfected B16 (<b>A,</b><b>B</b>) or Colon26 (<b>C,</b><b>D</b>) cells after treatment with etoposide (ETO) or control vehicle (DMSO). β-actin was the internal control. Data were quantitatively evaluated based on densitometric analysis (<b>B</b>, <b>D</b>). Results are fold-change compared with the level seen in SCR siRNA-treated B16 cells (<b>B</b>) or Colon26 cells (<b>D</b>), respectively. Data represent the mean ± SD. *, <i>P</i><0.05 (n = 3).</p

Attenuation of SLD5 expression results in marked DNA damage in MEFs.

<p>(<b>A</b>) Western blot analysis of SLD5 expression in WT and SLD5^+/− MEFs. β-actin was the internal control. (<b>B</b>) Quantitative evaluation of SLD5 expression as revealed in (<b>A</b>) based on densitometric analysis. Results are represented as fold-change compared with the level seen in WT MEFs. Data represent the mean ± SD. *, <i>P</i><0.05 (n = 3). (<b>C</b>) Western blot analysis of γ-H2AX expression in WT and SLD5^+/− MEFs after treatment with etoposide (ETO) or control vehicle (DMSO). β-actin was the internal control. (<b>D</b>) Quantitative evaluation of γ-H2AX expression as revealed in (<b>C</b>). Results are fold-changes compared with the level seen in WT MEFs treated with DMSO. Data represent the mean ± SD. *, <i>P</i><0.05 (n = 3).</p

Impaired cell cycle restoration after DNA damage in SLD5^+/− MEFs.

<p>MEFs were treated with DMSO (<b>A</b>) or etoposide (<b>B</b>) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110483#pone-0110483-g002" target="_blank">Figure 2</a> and the same number of living cells as indicated were cultured with fresh medium for 72 h. Cells were counted at the indicated time. Data represent the mean ± SD. *, <i>P</i><0.05 (n = 3).</p

Delayed and prolonged Rad51 expression after DNA damage by etoposide in SLD5^+/− MEFs.

<p>(<b>A</b>) Western blot analysis of Rad51 expression in WT and SLD5^+/− MEFs. β-actin was the internal control. MEFs were treated with etoposide for 12 h (−12∼0) and lysed at the indicated times. (<b>B</b>) Quantitative evaluation of Rad51 expression as revealed in (<b>A</b>) based on densitometric analysis. Results are fold-change compared with the level seen in WT MEFs before treatment with etoposide. Data represent the mean ± SD.*, <i>P</i><0.05 (n = 3).</p

The primers used in this study.

<p>The primers used in this study.</p

Tunicamycin inhibits JAK/STAT3 pathway downstream of gp130 independently of ER stress, PTP1B and SOCSs.

<p>Neonatal rat cardiac myocytes were cultured with the indicated concentrations of Tm for 8 hours. Total RNA was prepared and applied for reverse transcription. Real time PCR system was used to detect the mRNA expression of CHOP (A), Grp78 (A), PTP1B (C), SOCS1 and 3 (E) as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111097#s2" target="_blank">Material and Methods</a>’. The expression level of each gene was normalized with that of GAPDH, an internal control, and represented as value of fold induction relative to those of each non-treated group with Tm (control). Data were shown as mean ±S.D. (n = 3). **; <i>P</i><0.05 versus control at the multiple comparison test. After cardiac myocytes were pretreated with or without Tm (2 µg/mL) for 8 hours, cells were washed with medium and incubated for more 15 hours. Afterward, cells lysates were prepared for immunoblotting analyses with anti-CHOP, anti-Grp78 and GAPDH antibody. Experiments were repeated three times with similar results and representative data are shown in (B). Cardiac myocytes were pretreated with or without Tm (2 µg/mL) for 8 hours in the presence or absence of JTT551 (JTT), PTP1B inhibitor, and stimulated with LIF (300 U/I) for 15 minutes. Activations of STAT3 and ERK1/2 were analyzed by immunoblotting with anti-phospho-STAT3, anti-phopho-ERK1/2 and anti-GAPDH antibody. Representative images were shown in (D).</p

The combined treatment with IL-6 and sIL-6R fails to activate STAT3 in the presence of tunicamycin.

<p>Neonatal rat cardiac myocytes, pretreated with or without Tm (2 µg/mL) for 8 hours, were stimulated with IL-6 (20 ng/mL) plus sIL-6R (100 ng/mL) for 15 minutes. Activation of STAT3 and ERK1/2 were analyzed by immunoblotting with each phospho-specific antibody. Membranes were stripped and reprobed with anti-STAT3, anti-ERK1/2, or anti-GAPDH antibody, respectively. Representative images were shown (A). (B). For quantification, densitometric analyses for STAT3 or ERK1/2 phosphorylation were normalized with those of total STAT3 or total ERK, respectively. Values were converted based on that of each group treated with cytokine alone. Data were mean ± S.D. of three independent experiments. Dunnett test was performed for post-hoc multiple comparison test. *; <i>P</i><0.05 versus the combination of IL-6 and sIL-6R alone.</p

The Inhibition of N-Glycosylation of Glycoprotein 130 Molecule Abolishes STAT3 Activation by IL-6 Family Cytokines in Cultured Cardiac Myocytes

<div><p>Interleukin-6 (IL-6) family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130), a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK)1/2 through janus kinases (JAKs); however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm), an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF)-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs) and protein-tyrosine phosphatase 1B (PTP1B), which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R) could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.</p></div

Tunicamycin suppresses the gp130-mediated activation of JAK1 and 2.

<p>Neonatal rat cardiac myocytes, pretreated with the indicated concentrations of Tm for 8 hours, were stimulated with LIF (300 U/ml) for 15 minutes. The activation of JAK1 and JAK2 was analyzed by immunoblotting with the phospho-JAK1 and phospho-JAK2 specific antibodies. Membranes were stripped and reprobed with anti-JAK1, anti-JAK2, or anti-GAPDH antibody, respectively. Representative images were shown (A). (B). For quantification, densitometric analyses for JAK1 and JAK2 phosphorylation were normalized with those of total JAK1 or total JAK2, respectively. Values were converted based on that of each group treated with LIF alone. Data were mean ± S.D. of three independent experiments. Dunnett test was performed for post-hoc multiple comparison test. *; <i>P</i><0.05 versus LIF alone.</p