Fig. 4: FFSS reduces FAK activity, and FAK inhibitors mimic effect of FFSS.

a Protein expression of phospho-FAK (Y397), p-paxillin (Y118), and paxillin in Ocy454 cells exposed to FFSS for the indicated times. Phospho-FAK (Y397) was significantly decreased by FFSS within 10 min, and paxillin (the substrate of FAK) phosphorylation was decreased at later time points. b Dose responses of FAK inhibitors (PF562271 and VS-6063) on Ocy454 cells. FAK inhibitors were treated with indicated concentrations for 1 h. FAK inhibitors significantly reduced phospho-FAK (y-397) activity. Immunoblotting data are quantified in the bottom graph (n = 2 biologic protein replicates were performed). Data are shown with each measured value and best fit curve. c Control and HDAC4/5 compound mutant (H4H5-DKO) cells were treated plus/minus FAK inhibitors for 4 h followed by SOST RT-qPCR. H4H5-DKO cells fail to suppress SOST in response to FAK inhibitor treatment. (n = 4 RNA replicates for control, and n = 3 RNA replicates for H4H5-DKO were performed) P-values adjusted for multiple comparisons vs control are shown. d A volcano plot of RNA-seq analysis with FAK inhibitor, PF562271. RNA-seq analysis was performed with Ocy454 cells by vehicle and PF562271 treatment for 4 h. Significant genes were determined as log2FC > |1| and FDR < 0.05. e A Venn diagram of significantly changed genes among FFSS, FAK inhibitor (PF562271), and FAK gene deletion. FFSS was performed for 3 h with Ocy454 cells infected with a mock sgRNA-expressing lentivirus. Control uninfected Ocy454 cell were treated with FAK inhibitor for 4 h. FAK gene deletion was carried out by CRISPR-Cas9, and single-cell FAK-KO cell line was generated from bulk FAK-KO cell lines with puromycin selection. f A Pearson correlation matrix heatmap of RNA-seq analysis. Ocy454 cells were treated with/without FFSS, with/without FAK inhibitor (PF562271), and with/without FAK gene deletion by CRISPR-Cas9. One-way ANOVA followed by Tukey–Kramer post hoc test were used (c). Data are expressed as mean ± SEM. Each experiment was repeated three times (a, b). Source data are provided as a Source Data file.