Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Mar 8;10:25. doi: 10.1038/s41413-022-00204-1

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2022, corrected publication 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 8 — Plasma membrane depolarization induces preosteoblast senescence via the Ca²⁺/NFAT/ATF3/p53 signaling pathway. a Representative images of immunostaining of the voltage-gated calcium channel Ca_v1.2 in primary preosteoblasts. Scale bar, 20 μm. b Senescent ΔTsc1 and ΔRaptor preosteoblasts were treated with pinacidil (Pi) or KCl and incubated with the FluoForte probe to measure the relative cytosolic calcium levels. Calcium imaging data were quantified by normalization of the values to those of senescent wild-type (WT) preosteoblasts. Scale bar, 20 μm. c Representative Ca²⁺ traces for the senescent ΔTsc1 and ΔRaptor preosteoblasts with added pinacidil or KCl. d Immunostaining of NFATc1 and (e) western blot analysis of ATF3, p53, cyclin D1, and Rb phosphorylation levels in the senescent ΔTsc1 and ΔRaptor preosteoblasts. Scale bar, 10 μm in d. f Immunostaining of ATF3 and Osx in the ΔTsc1 and ΔRaptor mouse bone and quantitative analysis of the ATF3⁺ preosteoblasts (ATF3⁺ Osx⁺) relative to the total Osx⁺ preosteoblasts. Scale bar, 100 μm. g Western blot analysis of cyclin D1 expression and Rb phosphorylation in the ΔTsc1 preosteoblasts with interference with p53 expression. h Immunostaining of EdU in cells in g and quantitative analysis of EdU⁺ cells relative to total cells. Scale bar, 100 μm. i SA-β-gal staining of the cells in g and quantification of the proportion of SA-β-gal-positive cells in each population. Scale bar, 100 μm. j qPCR analysis of Cxcl1 and IL-6 mRNA in the cells in g. k Immunostaining of NFATc1 in the senescent ΔTsc1 preosteoblasts treated with BAPTA-AM (a calcium chelator) or left untreated. Scale bar, 10 μm. l Western blot analysis of ATF3 and p53 expression in the cells in k. m Immunostaining of EdU in the cells in k and quantitative analysis of EdU⁺ cells relative to total cells. Scale bar, 100 μm. n SA-β-gal staining of the cells in k and quantification of the proportion of SA-β-gal-positive cells in each population. Scale bar, 100 μm. o qPCR analysis of Cxcl1 and IL-6 mRNA in the cells in k. Data are shown as the mean ± SD. The numbers of samples (n) are indicated in each figure panel. P values were determined by two-tailed Student’s t test for single comparisons