Table 1.
The primers used in this study.
Figure 1.
Tunicamycin inhibits the glycosylation of gp130 completely, but does those of LIFR and IL-11Rα partially.
Neonatal rat cardiomyocytes were treated with the indicated concentrations of Tm for 8 hrs (A), or with 2 µg/mL of Tm for the indicated times (B), respectively. After each treatment, cell lysates were prepared and immunoblotted with anti-gp130, LIFR or IL-11Rα antibody, respectively. More than three independent experiments were performed with similar results and representative images were shown.
Figure 2.
Tunicamycin does not influence the localization of gp130 in cardiomyocytes.
Neonatal rat cardiomyocytes were treated with or without Tm (2 µg/mL) for 8 hours. Cultured cells were fixed and immunostained with anti-gp130 antibody or Hoechst for nuclei. Bar indicates 15 µm. Representative images were shown.
Figure 3.
Tunicamycin inhibits the activation of STAT3 and ERK by LIF or IL-11, respectively.
Cultured cardiomyocytes were pretreated with the indicated concentration of Tm for 8 hours, and stimulated with LIF (300 U/mL) (A) or IL-11 (20 ng/mL) (B) for 15 minutes. Activation of STAT3 and ERK1/2 was 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 and B). (C). For quantification, densitometric analyses for STAT3 or ERK1/2 phosphorylations 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. *; P<0.05 versus cytokine alone.
Figure 4.
Tunicamycin suppresses the gp130-mediated activation of JAK1 and 2.
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. *; P<0.05 versus LIF alone.
Figure 5.
Tunicamycin inhibits JAK/STAT3 pathway downstream of gp130 independently of ER stress, PTP1B and SOCSs.
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 ‘Material and Methods’. 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). **; P<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).
Figure 6.
The combined treatment with IL-6 and sIL-6R fails to activate STAT3 in the presence of tunicamycin.
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. *; P<0.05 versus the combination of IL-6 and sIL-6R alone.