Fig 2: Knockdown of RIOK3 significantly inhibits FAK activation in PDAC cells. Using lentiviral transfection, PANC-1 cells with stable knockdown of RIOK3 were created, followed by RNA extraction and sequencing. Up-regulated (red) and down-regulated (blue) mRNAs were characterized using volcano plots (A). Differential genes (| Log2FC | > 0.5, FDR ˂ 0.05) were used to perform a KEGG pathway enrichment analysis (B). The Log2 FC of all mRNA expression was used in GSEA (C). The spearman correlation coefficients of RIOK3 with all other genes in the TCGA-PDAC transcriptome database were used in GSEA (D). Western blot assay and quantitative analysis of the protein expression of RIOK3, FAK, Phospho-FAK(Tyr397), Phospho-FAK(Tyr925), Integrin 1, Integrin 4, and vinculin in cells (E). The qRT-PCR method was used to detect RIOK3 and FAK mRNA expression (F). Representative IHC staining of RIOK3 and Phospho-FAK(Tyr925) in pancreatic cancer tissues and correlation analysis between RIOK3 and Phospho-FAK(Tyr925) based on H-Score (G). R represents Pearson correlation coefficient. Analysis of the ratio of phospho-FAK/FAK in Figure 1E (H). The data shown represent the means (±SD) of biological triplicates. ∗p < 0.05, ∗∗p < 0.01, ANOVA test. The uncropped images of (E) were referred to in supplementary Figure 1.

Fig 3: High expression of Glutathione peroxidase 8 (GPX8) on cancer cells and CAFs promote lung cancer metastasis. BRD2/BRD4‐mediated upregulation of GPX8 promotes lung cancer metastasis by the regulation of p‐FAK and p‐Paxillin. GPX8‐mediated IL6 and CCL2 production of CAF may play crucial role for lung cancer metastasis

Fig 4: Phosphorylation of FAK and PI3K and cytoskeletal rearrangement in leptospire-infected cells.A: Phosphorylation of FAK in THP-1 and PI3K in HUVEC during infection with L. interrogans strain Lai for the indicated times; B: Quantification of immunoblotting bands reflecting FAK and PI3K phosphorylation in leptospire-infected THP-1 cells or HUVEC for the indicated times. Statistical data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. *: p < 0.05 vs the phosphorylation level (gray scale) of FAK or PI3K in the cells before infection; C: Leptospires in FAK- or PI3K-inhibited THP-1 and HUVEC after infection with L. interrogans strain Lai for 1 h. The blue plaques in the middle of cells indicate the nuclus. The red spots around the nuclus indicate the intracellular leptospires; D: Statistical summary of red fluorescence intensity reflecting the leptospires in FAK or PI3K-inhibited leptospire-infected cells. Statistical data from experiments such as shown in C. Bars show the means ± SD of three independent experiments. One hundred cells were analyzed for each of the specimens. *: p < 0.05 vs the red fluorescence intensity reflecting the leptospires in the FAK-uninhibited THP-1 or PI3K-uninhibited HUVEC before infection.

Fig 5: RIOK3 binds to and stabilizes the FAK protein. Co-IP assays were performed using RIOK3 antibody or IgG in protein lysates of PANC-1 cells and BxPC-3 cells, followed by western blot assays with RIOK3 and FAK antibodies (A). Co-IP assays were performed using FAK antibody or IgG in protein lysates of PANC-1 cells and BxPC-3 cells, followed by western blot assays with RIOK3 and FAK antibodies (B). A protein stability assay was used to test the stability of FAK protein in PANC-1-shRIOK3 cells (C). In HEK293T cells, HA-RIOK3 and Myc-FAK or Myc-FAK-Y925F were co-transfected, and the protein stability of Myc-FAK or Myc-FAK-Y925F was tested using a protein stability assay (D and E). The data shown represent the means (±SD) of biological triplicates. ∗p < 0.05, ∗∗p < 0.01, Student’s t-test (unpaired, two-tailed) or ANOVA test. The uncropped images of (A and B) were referred to in supplementary Figure 2 and the uncropped images of (C, D and E) were referred to in supplementary Figure 3.