Replication Data for: The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification

Objective: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). Methodology: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 microM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to view whether Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore’s ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. Results: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 microM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 microM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 microM Dynasore. Conclusion: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

目的：针对药物相关性颌骨坏死（medication-related osteonecrosis of the jaw）的治疗，一种潜在策略是使用局部制剂阻断此类药物进入口腔软组织。本研究旨在测试膦甲酸（phosphonoformic acid，PFA）与Dynasore的效能：前者是双膦酸盐通过特定钠依赖性磷酸盐共转运体（sodium-dependent phosphate contransporters，SLC20A1、20A2、34A1-3）进入细胞的抑制剂，后者为巨胞饮（macropinocytosis）抑制剂，二者均用于防止唑来膦酸诱导（zoledronate-induced，ZOL）的人牙龈成纤维细胞（human gingival fibroblasts，HGFs）死亡。

方法：采用MTT法剂量反应曲线确定PFA与Dynasore的非细胞毒性浓度。在50μM ZOL存在的条件下，评估经优化剂量的PFA和Dynasore恢复HGF活力的能力。通过定量实时逆转录聚合酶链反应（quantitative real-time RT-PCR）检测HGF中SLC转运体的表达水平。利用共聚焦荧光显微镜观察Dynasore是否抑制AF647标记的ZOL（AF647-ZOL）通过巨胞饮进入HGF。借助LysoSensor Green DND-189活细胞成像技术，测定Dynasore存在时的内体酸化程度。为进一步验证Dynasore干扰含ZOL内体成熟的能力，采用共聚焦荧光显微镜结合CellProfiler™软件分析图像，评估含AF647-ZOL或TRITC标记葡聚糖（对照）的成熟内体的核周定位情况。

结果：0.5mM PFA在72小时内未能挽救ZOL诱导的HGF活力下降，而10μM及30μM香叶基香叶醇（geranylgeraniol）可部分逆转该效应。与阳性对照组织相比，HGF中未检测到SLC转运体的表达。10μM Dynasore可完全阻止ZOL诱导的HGF活力丧失。在Dynasore存在时，AF647-ZOL与FITC标记葡聚糖在内体中呈现共定位。Dynasore可抑制内体酸化；30μM Dynasore能显著抑制含TRITC标记葡聚糖及AF647-ZOL的内体向核周区域的定位。

结论：Dynasore通过部分干扰巨胞饮过程，并抑制被认为是ZOL递送至细胞质所必需的内体成熟通路，从而有效防止ZOL诱导的HGF活力丧失。