Psuper retro puro vector

The knockdowns of Fascin and INF2-CAAX were achieved using pSUPER.Retro.puro vector (Oligoengine) encoding shRNA targeting mouse/human Fascin (Sun et al., 2011 (link), 2013 (link)), human Cortactin, and human INF2-CAAX (Korobova et al., 2013 (link)). To rescue Fascin knockdown or overexpress Fascin proteins, wild-type or mutant Fascin (S39E and 149-151A mutants) (Yang et al., 2013 (link)) cDNAs were subcloned into pLenti.CMV.blasticidin vector (Addgene_17486) (Campeau et al., 2009 (link)). pLKO vector encoding shRNAs for DRP1 were purchased from Sigma (sh1 TRCN0000318424; sh2 TRCN0000318425). The retroviral and lentiviral particles were packaged in HEK293 cells using the PEI transfection method, and concentrated as previously described (Yang et al., 2012 (link)).

RNA interference of STIM1 and Orai1 was performed using pSUPER.Retro.puro vector (Oligoengine) encoding shRNA. The target sequences were as follows: 5′-AGAAGGAGCTAGAATCTCAC-3′ (STIM1sh1), 5′-TCGGCCTGATCTTTATCGT-3′ (Orai1sh1), and 5′-CCAGCATTGAGTGTGTACA-3′ (Orai1sh2). To efficiently knockdown Orai1, two shRNAs targeting two different regions of the same gene were used simultaneously. In some experiments, two previously described STIM1 shRNA (sh2 and sh3, targeting 5′-GGCTCTGGATACAGTGCTC-3′ and 5′-GGATGCTGTCATTTTTTGA-3′; Yang et al., 2009 (link)) and a new Orai1 sh3 (targeting 5′-GGCTCTGGATACAGTGCTC-3′) were used to confirm STIM1 and Orai1 knockdown effects.

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.