Cytoplasmic PCNA interacts with β-actin and is necessary for the integrity of actin belt. (A) Interaction between PCNA and β-actin in RANKL-induced RAW264.7 cells. RAW264.7 cells with or without RANKL (100 ng/mL) induction for three days were used to perform co-IP assay using IgG and primary PCNA antibody. Western blot assay was then carried out to detect PCNA and β-actin in the immune complexes. Heavy chain: the heavy chains of control IgG and PCNA antibody. (B) The subcellular localization of PCNA and F-actin. RAW264.7 cells with or without RANKL (100 ng/mL) induction for three days were used to perform IF experiments. PCNA was labeled by Alexa 488 (green). F-actin was dyed with phalloidine-conjugated Alexa Fluor 594 (red). The images of the two bottom panels were two different representative osteoclasts. Scar bar: 5μm. (C) The effect of knockdown PCNA on the integrity of actin belt. RAW264.7 cells were transfected with siRNA targeting PCNA for 48 hours, then, the cells were treated with RANKL (100 ng/mL) for 3 days and IF assay were performed to assess the integrity of actin belt. Scar bar: 5μm. (D) The effect of PCNA knockdown on the expressions of β-actin and γ-actin. RAW264.7 cells were transfected with PCNA siRNA and treated with RANKL (100 ng/mL) for 3 days. Then, the cells were harvested and lysed for standard western blot assay using the indicated antibodies.