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

Version 1.0
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
Daniel Jones, 2024, "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", https://doi.org/10.48331/scielodata.QRF50O, SciELO Data, V1, UNF:6:je6g67yoz3qSMg4h+USlmw== [fileUNF]
Learn about Data Citation Standards.
Make Data Count (MDC) Metrics
since
0 Views
0 Downloads
0 Citations
Description
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. (2024-08-16)
Subject Medicine, Health and Life Sciences
Keyword Fibroblasts, Zoledronic Acid, Bisphosphonate-Associated Osteonecrosis of the Jaw
Related Publication Kirby J, Standfest M, Binkley J, Barnes C, Brow E, Cairncross T, et al. The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification. 2024;32:e2024-0224. doi: 10.1590/1678-7757-2024-0224
Notes Additional information about the dataset is found in the README.txt file.
License/Data Use Agreement

CC BY 4.0

Filter by
1 to 10 of 15 Files
Figure_1.jpg
Figure_1.jpg
JPEG Image - 110.3 KB
Published Aug 30, 2024
2 Downloads
MD5: 9186330d351f4128c47efd53f99678f4
MTT viability assay for determining the doses of ZOL (A), GGOH (B), and PFA (C) to use in rescue assays. (D) ZOL-induced viability loss rescue assay results where UN indicates untreated (set to 100% viability), ZOL indicates 50 microM ZOL. GG10 and GG30 indicate 10 microM and 30 microM of GGOH, respectively. Error bars represent SEM with the double asterisk ** or the plus + indicating p less than or equal to 0.01 and the triple asterisk *** or the hash # indicating p less than or equal to 0.001. Significant difference comparisons include panels A-C: doses significantly lower than their respective vehicle control and panel D: ZOL significantly lower than ZOL vehicle plus each of GG10 + ZOL and GG30 + ZOL significantly higher than ZOL.
Preview "Figure_1.jpg"
Figure_2.jpg
Figure_2.jpg
JPEG Image - 121.2 KB
Published Aug 30, 2024
2 Downloads
MD5: b7281b7dea20731da083ec90015eb7d3
Quantitative real-time RTPCR to determine the relative expression of selected SLC genes in HGFs with 18S rRNA gene normalization. The relative amount of (A) SLC20A1, (B) SLC20A2, (C) SLC34A1, (D) SLC34A2, and (E) SLC34A3 transporter gene expression in HGFs compared to the respective, known positive control tissue expression is shown. In each graph, HGF expression is set to one with positive control expression expressed as fold increase from that. Error bars represent standard error of the mean. Significant differences between HGF and positive control are denoted as follows: double asterisk ** indicates p less than or equal to 0.01, single asterisk * indicates p less than or equal to 0.05, and ns indicates non-significant difference.
Preview "Figure_2.jpg"
Figure_3.jpg
Figure_3.jpg
JPEG Image - 160.1 KB
Published Aug 30, 2024
2 Downloads
MD5: f90be0a594d0b2c8498d11115956d605
MTT viability assay to determine the toxicity of Dynasore in HGFs (A). (B) ZOL-induced viability loss rescue assay where UN indicates untreated (set to 100% viability) and the microM Dynasore concentrations are designated as Dyn 10, Dyn 30, etc. and ZOL is 50 microM in concentration. In panel B the corresponding combination of ZOL and Dynasore vehicles are designated as Combo 10 VEH, Combo 30 VEH, etc. Error bars represent SEM with triple asterisk *** or the plus + indicating p less than or equal to 0.001, double asterisk ** or the hash # indicating p less than or equal to 0.01, and single asterisk * indicating p less than or equal to 0.05. Significant difference comparisons include panel A: indicated doses significantly lower than their respective vehicle control. Panel B: ZOL significantly lower than ZOL VEH and each of the ZOL+ Dyn doses significantly higher than ZOL.
Preview "Figure_3.jpg"
Figure_4.jpg
Figure_4.jpg
JPEG Image - 136.2 KB
Published Aug 30, 2024
2 Downloads
MD5: 38ff2dbcf169524a0ac3ae289a7d7a82
Confocal fluorescence microscopy to determine whether dextran and ZOL colocalize in HGFs. From the same experiment, a representative photomicrograph of an HGF in the absence of Dynasore (A, C, E, and G) and a representative photomicrograph of an HGF in the presence of Dynasore (B, D, F, and H) in which exposure settings were kept constant. The red color is AF647-ZOL, the green is FITC-dextran, and the merged AF647-ZOL and FITC-dextran photomicrographs display the colocalizations highlighted by white arrowheads. The merged plus DAPI also includes the blue of the nucleus. 63X oil immersion objective, scale bar = 20 microm.
Preview "Figure_4.jpg"
Figure_5.jpg
Figure_5.jpg
JPEG Image - 238.1 KB
Published Aug 30, 2024
2 Downloads
MD5: 1848dacb3313d0438b270a286ed930bd
Confocal fluorescence microscopy of live HGFs exposed to LysoSensor Green DND-189 to measure endosomal acidification. Representative field of HGFs in the absence (A) and presence (B) of 30 microM Dynasore in which the exposure settings were kept constant. 20X objective, scale bar = 20 microm. (C) Image J quantitation of the average corrected total cell fluorescence (CTCF) where error bars represent SEM with double asterisk ** indicating p less than or equal to 0.01.
Preview "Figure_5.jpg"
Figure_6 .jpg
Figure_6 .jpg
JPEG Image - 111.2 KB
Published Aug 30, 2024
2 Downloads
MD5: a264a0113bfaeea8a79565c42aabc60d
Measurement of the perinuclear localization of ZOL and the positive control for macropinocytosis, dextran, in the absence and presence of 30 microM Dynasore. Violin plots display the HGF perinuclear proportion of intracellular TRITC-dextran (A) and AF647-ZOL (D) fluorescence. Horizontal red lines indicate the average perinuclear proportion of intracellular fluorescence from all cells analyzed per condition via CellProfiler™ software. The triple asterisks *** indicate p equal to or less than 0.001. Representative 63X oil immersion objective photomicrographs of TRITC-dextran intracellular distribution in the absence (B) and presence (C) of Dynasore. Representative 63X oil immersion objective photomicrographs of AF647-ZOL intracellular distribution in the absence (E) and presence (F) of Dynasore. Scale bar = 20 microm.
Preview "Figure_6 .jpg"
Graphical_Abstract.pdf
Graphical_Abstract.pdf
Adobe PDF - 84.1 KB
Published Aug 30, 2024
2 Downloads
MD5: 496fd3da516f74ba23d636c4a2e04675
Text summary and data displaying the major findings.
Preview "Graphical_Abstract.pdf"
HGFIMIRescue_DataGraph.jpg
HGFIMIRescue_DataGraph.jpg
JPEG Image - 66.2 KB
Published Aug 30, 2024
2 Downloads
MD5: db50edfaef672c4413910622875fe627
This graph displays the "data not shown" in the Dynasore rescue portion of Results. An optimized dose of imipramine (IMI) of 25 microM for one hour prior to 72 hours of incubation in the presence of zoledronate does not rescue the 50 microM zoledronate (ZOL)-treated HGFs. Error bars are SEM and the asterisk indicates ZOL-treated HGFs are significantly less viable than ZOL VEH control cells.
Preview "HGFIMIRescue_DataGraph.jpg"
HGFIMIRescue_PercentViabilityData.tab
Tabular Data - 1.9 KB
Published Aug 30, 2024
0 Downloads
11 Variables, 25 Observations UNF:6:cShYK+KhoIAqGIBEkEIfQQ==
This file contains the data used to make the graph that was "data not shown" in the Dynasore rescue portion of Results. The graph is the file entitled "HGFIMIRescue_DataGraph.jpg" and uploaded separately in the dataset. This graph shows that an optimized dose of imipramine (IMI) of 25 microM for one hour prior to 72 hours of incubation in the presence of zoledronate does not rescue the 50 microM zoledronate (ZOL)-treated HGFs. The corrected absorbance is the MTT assay 570 nm absorbance with the background 630 nm absorbance substracted. "MEDIA" control corrected absorbance was not statistically different than the ZOL vehicle (VEH) corrected absorbance; hence the ZOL VEH corrected absorbance was set to 100% viable and all other percent viabilities for the other conditions were expressed relative to the ZOL VEH viability. N=6.
Preview "HGFIMIRescue_PercentViabilityData.tab"
OsteoblastPFARescue_DataGraph.jpg
OsteoblastPFARescue_DataGraph.jpg
JPEG Image - 62.9 KB
Published Aug 30, 2024
2 Downloads
MD5: d3c779177990d91baf6a82fa93ff3a1e
This graph shows that optimized doses of phosphonoformic acid (PFA) from 0.25-0.75 mM for either a one-hour pre-treatment (PRE) prior or continual exposure during 72 hours of incubation in the presence of zoledronate do not rescue the 50 microM zoledronate (ZOL)-treated human calvarial osteoblasts. "MEDIA" control percent viability was not significantly different than the viability of the combined ZOL + PFA vehicles control (blue bar). All other percent viabilities for the other conditions were expressed relative to the ZOL + PFA VEHS' viability. Error bars are SEM and the asterisk indicates ZOL-treated human calvarial osteoblasts are significantly less viable than ZOL + PFA VEHS control cells. N=6.
Preview "OsteoblastPFARescue_DataGraph.jpg"
Edit File

This file has already been deleted (or replaced) in the current version. It may not be edited.

Restrict Access

Restricting limits access to published files. People who want to use the restricted files can request access by default. If you disable request access, you must add information about access to the Terms of Access field.

Learn about restricting files and dataset access in the User Guide.

Enable access request
You must enable request access or add terms of access to restrict file access.
Save Changes
Edit Embargo

The selected file or files have already been published. Contact an administrator to change the embargo date or reason of the file or files.
Delete Files

The file will be deleted after you click on the Delete button.

Files will not be removed from previously published versions of the dataset.

Select File(s)

Please select one or more files.

Share Dataset

Share this dataset on your favorite social media networks.

Dataset Citations

Citations for this dataset are retrieved from Crossref via DataCite using Make Data Count standards. For more information about dataset metrics, please refer to the User Guide.

Sorry, no citations were found.
Restricted Files Selected

The selected file(s) may not be downloaded because you have not been granted access.

Download Options

The files selected are too large to download as a ZIP.

You can select individual files that are below the 100.0 MB download limit from the files table, or use the Data Access API for programmatic access to the files.

Select File(s)

Please select a file or files to be downloaded.

Restricted Files Selected

The restricted file(s) selected may not be downloaded because you have not been granted access.

Click Continue to download the files you have access to download.

Delete Dataset

Are you sure you want to delete this dataset and all of its files? You cannot undelete this dataset.

Delete Draft Version

Are you sure you want to delete this draft version? Files will be reverted to the most recently published version. You cannot undelete this draft.

Unpublished Dataset Private URL

Private URL can only be used with unpublished versions of datasets.

Unpublished Dataset Private URL

Are you sure you want to disable the Private URL? If you have shared the Private URL with others they will no longer be able to use it to access your unpublished dataset.

Delete Files

The file(s) will be deleted after you click on the Delete button.

Files will not be removed from previously published versions of the dataset.

Compute

This dataset contains restricted files you may not compute on because you have not been granted access.

Deaccession Dataset

Are you sure you want to deaccession? This is permanent and the selected version(s) will no longer be viewable by the public.

Deaccession Dataset

Are you sure you want to deaccession this dataset? This is permanent an it will no longer be viewable by the public.

Version Differences Details

Please select two versions to view the differences.

Version Differences Details
 
Version:
Last Updated:
Version:
Last Updated:
Select File(s)

Please select a file or files for access request.

Select File(s)

Embargoed files cannot be accessed. Please select an unembargoed file or files for your access request.

Edit Tags

Select existing file tags or create new tags to describe your files. Each file can have more than one tag.

Request Access

  You need to Log In to request access.

Dataset Terms

This dataset is made available under the following terms. Please confirm and/or complete the information needed below in order to continue.

Our Community Norms as well as good scientific practices expect that proper credit is given via citation. Please use the data citation shown on the dataset page.

CC BY 4.0

Preview Guestbook

Upon downloading files the guestbook asks for the following information.

Account Information

Package File Download

Use the Download URL in a Wget command or a download manager to download this package file. Download via web browser is not recommended. User Guide - Downloading a Dataverse Package via URL

https://data.scielo.org/api/access/datafile/

Request Access

Please confirm and/or complete the information needed below in order to request access to files in this dataset.

Compute Batch
Clear Batch
Dataset Persistent Identifier Change Compute Batch
Compute Batch
Submit for Review

You will not be able to make changes to this dataset while it is in review.

Publish Dataset

Are you sure you want to republish this dataset?

Select if this is a minor or major version update.

Publish Dataset

This dataset cannot be published until Journal of Applied Oral Science is published by its administrator.

Publish Dataset

This dataset cannot be published until Journal of Applied Oral Science and SciELO Data are published.

Return to Author

Return this dataset to contributor for modification. The reason for return entered below will be sent by email to the author.