Sc 437282

The recombinant human Semaphorin 3A was purchased from R&D systems (Abingdon, UK). We used the human KHOS, MNNG/HOS, Saos-2 and MG-63, and mouse POS-1, MOS-J and K7M2 osteosarcoma cells (ATCC, Manassas, VA, USA). The mouse MC3T3 cells were used as a representative of osteoblasts and RAW 264.7 as macrophage-like, pre-osteoclast cells (ATCC, Manassas, VA, USA). Tissue culture medium (DMEM and alpha-MEM) was obtained from Gibco, Thermofisher (Leicestershire, UK). All primary antibodies were purchased from Cell Signalling Biotechnology (MA, USA) except rabbit anti-Semaphorin3A was purchased from Abcam (Abingdon, UK) and rabbit anti-actin was obtained from Sigma-Aldrich (Dorset, UK). Mouse macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK) and receptor activator of NFκB ligand (RANKL) was a gift from Patrick Mollat (Galapagos SASU, France), and was prepared as previously described^{44 (link)}. Overexpression of Sema3A was achieved by use of control (sc-437282) and Sema3A lentiviral activation particles (sc-400716-LAC) Santa-Cruz (Heidelberg, Germany).

Cloning the MALT1-overexpressed DU145 cells was performed using MALT1 CRISPR/dCas9 lentiviral activation transduction particles, a synergistic activation mediator (SAM) transcription activation system for the transcriptional activation of endogenous genes, according to the manufacture’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, DU145 cells were plated onto 6-well plates 24 h before transfection. The culture media was replaced with RPMI-1640 medium plus 10% FCS and 5 µg/mL polybrene (Santa Cruz Biotechnology), then transduced with MALT1 lentiviral activation transduction particles (sc-400791-LAC-2, Santa Cruz Biotechnology). Two days after transduction, the cells were selected by incubating with 10 µg/mL puromycin dihydrochloride for at least three generations. The mock-transfected cells were transduced with control lentiviral activation particles (sc-437282, Santa Cruz Biotechnology) and clonally selected in RPMI-1640 medium plus 10% FCS and 10 µg/mL puromycin dihydrochloride.

THP-1 monocytes were plated at 2 × 10⁵ cells/mL in 12-well plates overnight. A final concentration of 1 uM PMA was added the following morning, and THP-1 cells were allowed to differentiate for 72-h. Non-adherent cells were aspirated following 72-h exposure and replaced with fresh media containing 10 ug/mL polybrene (Catalog# sc-134220A, Santa Cruz Biotechnology). THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology). THP-1 macrophages were spun in the presence of 200 uL activation particles / well at 300 × g for 45 min. polybrene-containing media was replaced with fresh media 8-h post transduction. Confirmation of positive lentiviral transduction of THP-1 macrophages was monitored by GFP visualization and THP-1 macrophages harvested 72-h following transduction. Total and small RNA fractions were purified using the mirVana PARIS RNA and Native Protein Purification kit (Catalog# AM1556, Invitrogen) following standard protocols.