Figure 6. Effects of CLU silencing on TNFα and ROS levels in cultured OA chondrocytes. (A) Representative immunocytochemical images and evaluation of TNFα+ cytospinned OA-HACs after 72 hours of CLU silencing. (B) Basal mRNA expression of NOX isoforms by RT-PCR in cultured chondrocytes. (C, D) RT-PCR and blot analysis of NOX4 expression in scramble and CLU-silenced OA-HACs. (E, F) Representative images and quantitative measurement of ROS levels (green fluorescence) after 12, 24 and 48 hours of CLU silencing compared with control (scramble) and HACs from fractured patients; original magnification, 100X. Data are expressed as mean values ± SEM of three independent experiments (each using pooled samples from n=3 donors) performed in triplicate. Abbreviations: F-HACs, fractured patient human articular chondrocytes; OA-HACs, OA-Human articular chondrocytes; ADU, arbitrary densitometric unit; F.I., fluorescence intensity. Student’s t-test: *P < 0.05, **P < 0.01 and ***P < 0.001.