Figure 2.

circPDE4D Knockdown Induced Matrix Degradation in Chondrocytes

(A) HCs were stably transfected with three different circPDE4D shRNAs or the negative control. The expression of circPDE4D and linear PDE4D was evaluated by qRT-PCR. (B) The cells were stably transfected with sh-circPDE4D #1, #2, or #3 or the negative control. Representative images of Alcian blue (SW1353) and toluidine blue staining (HCs) were shown. (C) The mRNA levels of matrix components Aggrecan, Col2a1, and SOX9 in HCs transfected with sh-circPDE4D #1, #2, #3 or negative control were evaluated by qRT-PCR. (D) The mRNA levels of catabolic enzymes MMP3, MMP13, ADAMTS4, and ADAMTS5 in HCs transfected with sh-circPDE4D #1, #2, #3 or negative control were evaluated by qRT-PCR. (E) The protein expression of Aggrecan, Col2a1, SOX9, MMP3, MMP13, ADAMTS4, and ADAMTS5 in sh-circPDE4D-treated HCs and control chondrocytes was determined by western blotting. (F) sh-circPDE4D-treated or vector control-treated HCs were stimulated with various inflammatory factors (IL-1β, IL-6, and TNF-α) or cultured under certain metabolic stress conditions (serum deprivation, glutamine deprivation, glucose deprivation, or oxidative stress). The histogram demonstrates the relative changes in Aggrecan expression compared with the matched control. The data were obtained from three independent experiments with three independent donors (presented as the means ± SDs; A, C, D, and F) or were representative of three independent experiments with similar results (B and E). ∗p < 0.05 and ∗∗p < 0.01 versus the control or indicated group. The data were analyzed using two-tailed t tests (A, C, D, and F).