Figure 5. Effects of CLU silencing on viability, proliferation/death and differentiation of cultured OA chondrocytes. (A) CLU mRNA and protein expression after 48 and 72 hours, respectively, in scramble (control) and siCLU transfected OA-HACs. (B) Cell viability by MTT assay after 24, 48 and 72 hours of scramble and siCLU transfected OA-HACs. (C) Representative immunocytochemical images and bar graphs showing Ki67+ and p21+ nuclei in cultured OA-HACs and (D) cleaved caspase-3+ cytospinned OA-HACs after 72 hours of silencing. (E) Gene array shows the modulation of NFkB and Stat-3-related genes in siCLU transfected OA-HACs compared with scramble after 48 hours of silencing. Transcript levels were normalized on GAPDH (internal reference gene). (F) mRNA levels by Real-Time PCR of COL2A1, ACAN, MMP13, COL10A1, and (G) representative blot and densitometric analysis of MMP13 and COL10A1 in scramble and CLU-silenced OA-HACs. Data are expressed as mean values ± SEM of three independent experiments (each using pooled samples from n=3 OA donors) performed in triplicate. Abbreviations: OA-HACs, OA-Human articular chondrocytes; OD, optical density unit; RLU, relative light unit. Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.