Pthrp

Mouse osteocytic MLO‐Y4 cells (generously donated by Dr. Lynda Bonewald) were grown in α‐MEM supplemented with 2.5% fetal calf serum (FCS) and 2.5% fetal bovine serum (FBS). Murine monocyte cell line (RAW 264.7) was purchased from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. All cells were cultured with penicillin (100 units/ml) and streptomycin (100 μg/ml) in a 5% CO₂ humidified incubator at 37°C. MLO‐Y4 cells were plated at 5 × 10⁵ cells/cm² on collagen‐coated glass slides (FlexCell); the next day, fresh medium with 1% FBS was added for 24 h. Then, cells were submitted or not (static control, SC) to mechanical stress by laminar FF with a shear stress of 10 dynes/cm² for 10 min in a Flexcell® Streamer® shear stress device. The media flowed over the cells was consistently serum‐depleted α‐MEM in every experimental condition. Or they were stimulated by PTHrP (1‐37) (Bachem) for 10 min. Cells were cultured with fresh medium (α‐MEM without serum) for an additional 18 h after being stimulates with PTHrP (1‐37), FF or SC to collect the cell conditioned medium (CM) in the different experimental groups. RNA was isolated after 6 h of the stimulation with PTHrP (1‐37),  FF, or SC.

Tumor cells were isolated from transgenic mammary tumors as previously described. Briefly, dissected tumors were minced into fragments under sterile conditions and subjected to enzymatic digestion with Collagenase-Type3 (Worthington, Cat#: LS004183) at 2 mg/ml, Dispase (Gibco, Cat#: 17105-041) at 2 mg/ml, Gentamycin (Gibco, Cat#:15710-064) at 50 µg/ml, Amphotericin B (Sigma, Cat#:A2942) at 250 µg/ml, and 5% FBS in DMEM/F12 media for 3 h with intermittent shaking. Following digestion, tumor organoids were pelleted and then treated with NH4Cl (Stem Cell Technologies, Cat # 07800) to lyse RBCs, following which, the pellet was washed three times with PBS. Organoids were then passed through a 70 µm cell strainer, counted and used for transplantation experiments or cultured at a density of 3 × 10⁶ cells/55cm². Proliferation of cultured cells was measured by assessing BrdU incorporation (Cell proliferation ELISA Kit 11647229001; Roche) after addition of Dox (2 µg/ml) or PTHrP (Bachem, Cat# 4017147.0500) to the culture media.