Fenoldopam mesylate

MLO-Y4 osteocytes were cultured on collagen I-coated dishes in MEMα (Life Technologies) supplemented with 5% fetal bovine serum (FBS), 5% calf serum (CS), and 1% penicillin/streptomycin (P/S) at 37°C and 5% CO₂. Osteocytes were seeded at 2800 cells/cm² for 72 hours before application of fluid flow. MC3T3 osteoblasts were cultured in MEMα supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Osteoblasts were seeded at 2000 cells/cm² 72 hours before receiving conditioned media. Osteocytes were treated with 10 μM fenoldopam mesylate (Sigma), 5 μM tubastatin (Sigma) – an HDAC6 inhibitor that we have previously reported increases the stiffness of primary cilia to reduce osteocyte mechanosensitivity ^{(39 )}–, or vehicle control (<0.1% DMSO) for 16 hours prior to experimentation, as previously performed ^{(30 )}.

MLO-Y4 osteocytes were cultured on collagen I-coated (Corning) dishes in MEMα (Life Technologies) supplemented with 5% fetal bovine serum, 5% calf serum, and 1% penicillin/streptomycin at 37°C and 5% CO₂. Fenoldopam mesylate (Sigma) was used at 10 μM diluted in DMSO, dimethyl sulfoxide, (Sigma) and normal culture media, as previously described (Kathem et al., 2014 (link); Upadhyay et al., 2014 (link)). Lithium chloride (Sigma) was used at 500 μM diluted in normal culture media – a dose response from 50 μM to 10 mM was examined with 500 μM being the lowest dose to increase length significantly, data not shown. These agents, or their vehicle control, were applied to cells for 16 hours prior to experimentation. MTT, methylthiazolyldiphenyl-tetrazolium bromide, assay (Sigma) was performed according to manufacturer’s protocol to assess cell viability with drug treatments. Phase contrast microscopy with an Olympus CKX41 inverted microscope and 40× objective was used to assess cell morphology.