Fig. 3: FAK is required for FFSS-induced gene expression changes in osteocytes.

a Left, Sost mRNA expression was assessed after treatment with FAK inhibitors (PF562271, VS-6063, and PF431396) for 4 h (n = 2 biologic replicates were performed). All FAK inhibitor treatments reduced Sost mRNA expression in Ocy454 cells grown in static conditions. Right, cell viability was measured after 4 h treatment with the indicated FAK inhibitors (n = 4 biologic cell replicates were performed). Data are shown with each measured value and best fit curve. b Single-cell FAK-KO cells were generated by CRISPR-Cas9. Protein expression of FAK, p-paxillin (Y118), and paxillin in control and FAK gene deleted (FAK-KO) Ocy454 cells. Phospho-paxillin was significantly decreased in FAK-KO cells. Each experiment was independently repeated three times. c, d Control and FAK-KO cells were as indicated for 3 h followed by RT-qPCR analysis for Sost. For PTH treatments, cells were treated under static condition at a PTH 1–34 concentration of 242 nM. While basal Sost levels are reduced in FAK-KO cells, there is no further decrease in response to FFSS. In contrast, PTH further suppresses SOST in FAK-deficient cells. (n = 4 biologic cell replicates were performed for RNA analysis) P-values adjusted for multiple comparisons are shown versus STATIC condition in control cells. e RNA-seq analyses were performed with control and FAK-KO cells treated for 3 h with/without FFSS. Volcano plots representing the effects of FFSS in each cell type (control and FAK-KO) are shown. Differentially expressed genes (DEGs, log2FC > 1 or < −1, FDR < 0.05) are shown as blue points. While many DEGs are noted in response to FFSS in control cells, very few FFSS-induced DEGs are detected in FAK-KO cells. f Genes whose expression was significantly changed by FFSS in control and FAK-KO cells. g Most FFSS-regulated gene expression changes require the presence of FAK. Heatmap of expression values for DEGs, shown as Z scores of log2(CPM) values for a given gene across all samples. Rows, genes; columns, samples of control and FAK-KO single-cell clones. One-way ANOVA followed by Tukey–Kramer post hoc test was used (c, d). Data are expressed as mean ± SEM. Source data are provided as a Source Data file.