Ibandronate

Cells were cultivated in cell culture flasks at 37°C and 5% CO₂. The culture media were as recommended by the American Type Culture Collection (ATCC) for MDA-MB-231 breast cancer DMEM (Sigma-Aldrich, St. Louis, MO, USA), which contained 10% fetal calf serum (FCS); PC-3 prostate carcinoma DMEM-F12 (Sigma-Aldrich) with 10% FCS. MG-63 and U2-OS osteosarcoma were cultured in AlphaMEM (Biochrom, Berlin, Germany) medium containing 10% FBS. For the HMC1.1 cell line, we used Iscove's Modified Dulbecco's Medium (IMDM; Thermo Fisher Scientific, Waltham, MA) supplemented with 260 nM thioglycerol (Sigma-Aldrich) and 20% fetal bovine serum (FBS). All culture media contained 10 μg/mL gentamycin (Sigma-Aldrich). To guarantee optimal growth, cells were split two times a week and reseeded at a density of 2−5 × 10⁵ cells/mL.
One day after splitting, 32 μM simvastatin (Sigma-Aldrich) or 150 μM ibandronate (Sigma-Aldrich) were added to the culture medium for 72 hours. This is the dose that attenuated cell proliferation with a half maximal effect (EC50) (data not shown).
NADP/NADPH analyses were performed directly in 96-well culture plates after 24 or 48 hours, according to the manufacturers' instructions of the NADP/NADPH Glo Assay (Promega, Madison, WI, USA).

Zoledronate used in in vitro experiments was kindly provided by Novartis Pharma (Basel, Switzerland). Clodronate, Pamidronate, Ibandronate were purchased from Sigma Aldrich (St. Louis, MO, USA), as well as Zoledronate used in in vivo studies. The structure of bisphosphonates is depicted in Figure 1. Bisphosphonate stock solutions were prepared in distilled water, adjusted to pH 7.4 and filter-sterilized prior to use. Empty liposomes and Clodrolip were prepared as described before [43] (link). Other reagents were purchased from Sigma Aldrich. All solutions were prepared in distilled water and filter-sterilized prior to use.

Type I collagen extracted from rat-tail, Monocrotaline (MCT) and Ibandronate (IB) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Collagenase II was purchased from Biosharp (Shanghai, China). The Rac1 Activation Assay Biochem Kit™ was acquired from Cytoskeleton (Denver, CO, USA). EGM™-2 BulletKit™ was obtained from Lonza (Walkersville, MD, USA). Anti-FDPS was purchased from Abcam (Cambridge, MA, USA). GAPDH as the loading control was purchased from MultiSciences (Hangzhou, China). Other unspecified antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Plates with different concentrations of fluvastatin (brand Lescol; Novartis) were made according to the protocol described in previous studies (Mörck et al. 2009 (link); Rauthan et al. 2013 (link)). Additional compounds used in this study were: rosuvastatin (Crestor; AstraZeneca); mevalonolactone (Sigma); ibandronate (Sigma); paraquat (Sigma); and NAC (Sigma). These were dissolved in water except for rosuvastatin, which was dissolved in DMSO.