Fig 1.
TGF-β1 up-regulates CTGF expression in human granulosa cells.
(A) SVOG cells were treated with 5 ng/mL TGF-β1, and the mRNA levels of CTGF were analyzed at different time points by RT-qPCR. The level of CTGF mRNA at each time point was normalized to the GAPDH mRNA level at the same time point. (B) SVOG cells were treated with 5 ng/mL TGF-β1 for 3, 6 and 12 hours, and the protein levels of CTGF were examined by western blot. The level of CTGF protein at each time point was normalized to the β-tubulin protein level of 3 hour control. (C) SVOG cells were transfected for 48 hours with 50 nM control siRNA (si-Ctrl) or CTGF siRNA (si-CTGF) and then treated for 3 hours with 5 ng/mL TGF-β1. The protein levels of CTGF were examined by western blot. (D) Primary human granulosa cells were treated with 5 ng/mL TGF-β1 for 3 hours, and the protein levels of CTGF were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter were significantly different (p<0.05).
Fig 2.
The TGF-β receptor is required for the TGF-β1-induced up-regulation of CTGF expression in SVOG cells.
(A) and (B) SVOG cells were pretreated with 10 μM SB431542 for 1 hour and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (A) and protein (B) levels of CTGF were examined by RT-qPCR and western blot, respectively. (C) and (D) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or TβRI siRNA (si-TβRI) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (C) and protein (D) levels of CTGF and TβRI were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter were significantly different (p<0.05).
Fig 3.
Smad signaling pathway is required for the TGF-β1-induced up-regulation of CTGF expression in SVOG cells.
(A) and (B) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or Smad4 siRNA (si-Smad4) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (A) and protein (B) levels of CTGF and Smad4 were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter were significantly different (p<0.05).
Fig 4.
Knockdown of Smad2 or Smad3 attenuates TGF-β1-induced up-regulation of CTGF expression in SVOG cells.
(A) and (B) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or Smad2 siRNA (si-Smad2) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (A) and protein (B) levels of CTGF and Smad2 were examined by RT-qPCR and western blot, respectively. (C) and (D) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or Smad3 siRNA (si-Smad3) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (C) and protein (D) levels of CTGF and Smad3 were examined by RT-qPCR and western blot, respectively. (E) and (F) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or Smad2 plus Smad3 siRNAs (si-Smad2+3) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (E) and protein (F) levels of CTGF, Smad2 and Smad3 were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter were significantly different (p<0.05).
Fig 5.
The ERK1/2 signaling pathway is required for the TGF-β1-induced up-regulation of CTGF expression in SVOG cells.
(A) and (B) SVOG cells were pretreated with 10 μM U0126 for 1 hour and then treated with 5 ng/mL TGF-β1 for 3 hours. The mRNA (A) and protein (B) levels of CTGF were examined by RT-qPCR and western blot, respectively. (C) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or ERK1/2 siRNAs (si-ERK1/2) for 48 hours and then treated with 5 ng/mL TGF-β1 for 3 hours. The protein levels of CTGF and ERK1/2 were examined by western blot. (D) SVOG cells were transfected with 50 nM control siRNA (si-Ctrl) or Smad4 siRNA (si-Smad4) for 48 hours and then pretreated with U0126 (10 μM) for 1 hour. After pretreatment, the cells were treated with 5 ng/mL TGF-β1 for 3 hours. The protein levels of CTGF were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter were significantly different (p<0.05).