Short hairpin RNA introduction by retroviral gene transfer. The retroviral vectors pSuper-sh-Cas puro and pSuper-sh-RelA puro were cloned with the mouse Cas target sequence 5′-GCATGACATCTACCAAGTT-3′ and the mouse Rela target sequence 5′-GAAGAAGAGTCCTTTCAAT-3′, respectively, into a pSuper retro puro vector (Oligoengine, Seattle, WA). For double gene silencing of Cas and RelA, the retroviral vector pSuper-sh-RelA puro, in which the mouse Rela target sequence was cloned into a pSuper retro puro vector, was used together with pSuper-sh-Cas hygro. The plasmid harboring the retroviral construct was transiently transfected into the packaging cell line HEK293T or Plat-E (respectively provided by H. Ichijo and T. Kitamura, The University of Tokyo). Supernatants containing retroviral particles were collected 24 to 48 hours after transfection and used immediately to infect cell cultures. Subconfluent MLO-Y4 osteocytes were exposed to viral supernatants in the presence of polybrene (6 μg/ml) for 24 hours and then incubated in fresh culture medium for 2 days. Infected cells were selected by culturing them in the presence of puromycin (5 μg/ml) for at least 4 days. For double gene silencing, doubly infected cells were selected by culturing them in the presence of hygromycin (400 μg/ml) and puromycin.

Small interfering RNA transfection. To silence the endogenous expression of Cas in HEK293 cells, Cas-targeted small interfering RNA (siRNA) (Stealth RNAi, BCAR1-HSS114273, Thermo Fisher Scientific) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were used for experiments. Similar results were obtained using another Cas-targeted siRNA (Stealth RNAi, BCAR1-HSS114272, Thermo Fisher Scientific).

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