Fig 2: The identified humoral antigens in the focal adhesion complexes are ribonucleoproteins (RNPs) colocalized with ribosomal translation machinery.a GO enrichment analysis of the five protein antigens correlated with focal adhesion complexes. b Fluorescent staining of single-strand nucleotides using acridine orange in MKN1 cells. The blue color represents Hoechst nuclear staining. DIC, differential interference contrast image. c Fluorescent dual staining of anti-FAK antibody (red) and Cy5-conjugated oligo-dT probe (green) in human gastric cancer cells MKN1 and GSU, a lung adenocarcinoma cell A549, and a pancreatic cancer cell PANC-1. The blue color represents Hoechst staining. The white bars indicate 20 µm. d Fluorescent immunocytochemistry of FAK (red) and a ribosome marker (ribosomal protein S6 [RPS6]) (green). The blue represents nuclear staining. The white bars indicate 20 µm. b–d White arrowheads indicate representative colocalization signals, and the similar colocalizations were reproducibly observed in all analyzed cells with formations of visible focal adhesions. e The validity of the anti-FAK antibody in our RIP procedure was confirmed by immunoblots. The arrowhead and asterisk represent the FAK protein and heavy chain of the anti-FAK rabbit antibody, respectively. f The left and right panels show electropherograms of the precipitated RNA and constructed cDNA sequencing library, respectively, of FAK-RIP experiments in biological duplicates (#1 and #2). g Plots of the results of the next-generation sequencing of FAK-RIP. The x and y-axes represent the numbers of sequencing reads of input cellular RNA and the minimum of those in the two FAK-RIP experiments, respectively. Each dot represents each transcript/gene, while red dots indicate the enriched transcripts/genes used in the motif enrichment analysis. h The top five enriched motifs in the transcript sequences among FAK-RIP-enriched genes (354 genes) compared to FAK-RIP-diminished genes (454 genes). i Histograms of the numbers of genes plotted according to the numbers of “CAGCCC” motifs within the gene transcript sequences. The left and right panels represent FAK-RIP-enriched and -diminished gene sets, respectively.

Fig 3: Focal adhesion-related protein complexes are major and common humoral antigens in tumor microenvironments.a GO enrichment analysis of the eight identified protein antigens. b–i Fluorescent immunocytochemistry of the identified protein antigens (green) along with focal adhesion kinase (FAK) (red) onto a human gastric cancer cell line MKN1. The blue color represents Hoechst nuclear staining. The white bars indicate 20 µm. The white arrowheads indicate representative hot spots of colocalization between protein antigens and focal adhesions (only in b–f). Similar colocalizations were reproducibly observed in all analyzed cells with formations of visible focal adhesions (only in b–f). The same immunocytochemical staining was also performed onto another human gastric cancer cell line GSU, obtaining similar results (Supplementary Fig. 4). j Co-immunoprecipitation experiments of the FAK complex. The upper and lower panels at the left side show co-IP using anti-FAK antibody for MKN1 and anti-GRB7 antibody for GSU, respectively, followed by immunoblots using the indicated antibodies. The right-side panels show co-IP using anti-FAK antibody for A549, a lung adenocarcinoma cell line, and HEK293 transfected with GRB7-myc/His construct, respectively, followed by immunoblots using the indicated antibodies. Co-IP experiments were conducted at least twice.

Fig 4: Western blot and quantification of pFAK and FAK expressions of hPDLs cultured in CS0, CS0.5, and CS1. Data presented as mean ± SEM, n = 6 for each group. Quantification of Western blot bands expressed as pFAK/FAK ratios. A p-value of <0.05 was considered as a significant difference according to the calculation of Scheffe’s multiple comparison test. * represents significant differences when compared to CS0 and # represents significant differences when compared to CS0.5.

Fig 5: TAT-Cx43266-283 Reduces Y576 and Y577 FAK Phosphorylation in GSCsPrimary G9 (A) or G166 (B) GSCs were incubated with TAT or TAT-Cx43266-283. Western blot analysis for FAK, Y576 FAK, and Y577 FAK. The results were normalized with their corresponding TAT (assigned a value of 1) and are the means ± SEM of at least four independent experiments (***p < 0.001, **p < 0.01, *p < 0.05; TAT-Cx43266-283 versus TAT, t test). See also Figure S2.