Fig 2: Schematic depiction of the proposed model. CP generates DNA damage, which eventually leads to cell apoptosis. Meanwhile, RAP1 is upregulated after CP treatment, possibly through a direct or indirect induction by DNA damage response. The cytoplasmic fraction of RAP1 thus acts to facilitate the IKK-mediated activation of NF-κB signaling, which subsequently induces transcription of downstream factors, including the apoptosis inhibitor BCL-2. Therefore, RAP1 functions through activating NF-κB signaling to mediate resistance to CP

Fig 3: Proliferation of NSCLC cells is inhibited after Rap1 deletion. (a) RAP1 mRNA expression in 16HBE and HSAEC1-KT (HSA for abbreviation in following) lung epithelial cells and A549, PC9 and HCC827 NSCLC cells measured by qRT-PCR. (b) Western blotting analysis for RAP1 in cytoplasmic and nuclear fractions of lung epithelial and NSCLC cells, representatives of three independent experiments. (c) Immunofluorescence (IF) detection of RAP1 (red) in 16HBE and NSCLC cells. Arrowhead: cytoplasmic RAP1; scale bar: 5 μm. (d) RAP1 mRNA expression in A549, PC9 and HCC827 cells after transducing lentiviral vector coding two shRNAs against RAP1 (shRAP1) or scramble shRNA. (e) Proliferation of NSCLC cells after transduction with shRAP1 or scramble shRNA. Cells were plated in 96-well plates, cultured for 96 h and viability determined through cell counting kit-8 (CCK-8) assay. (f) Colony-formation assays for A549 cells after transduction with shRAP1 or scramble shRNA, detected by Giemsa staining. Statistics were generated from three independent experiments. **P<0.01 ***P<0.001, Student’s t-test (two groups) or one-way analysis of variance with Bonferroni’s correction (three or more groups); error bar: ±S.D.

Fig 4: Cytoplasmic RAP1 is an indicator of high-grade NSCLC. (a and b) Expression of cytoplasmic and nuclear RAP1 in lung adenocarcinoma (a) and squamous cell carcinoma (b) tissues, as well as peri-tumoral normal tissues (PTNT). (c and d) Expression of cytoplasmic RAP1 in Grade I–III adenocarcinoma (c) and squamous cell carcinoma (d) tissues. (e) Kaplan–Meier analysis and Log-rank (Mantel–Cox) test of the survival between RAP1-high (n=33, expression >0.4) and RAP1-low (n=59, expression <0.4) adenocarcinoma patients. (f) Representative images of RAP1 expression in adenocarcinoma, squamous cell carcinoma and PTNT, arrowhead: cytoplasmic RAP1; scale bar: 10 μm. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test

Fig 5: Human RAP1, GFAPɛ and PS1 interact with one another in vitro. a) Proteins were expressed in BL21 and BL21(DE3) cells. MBP-PS11–85 and the His6-tagged GFAP proteins (amino acids 204—end) were purified prior to being added to glutathione beads alone or beads prebound with GST-RAP1. The input lanes show the purified proteins (lanes 1, 3, and 4) or GST-RAP1 lysate (lane 2). Eluates from the glutathione beads are in lanes 5 through 13. Proteins included in each sample are listed above the blot image. The blot was probed simultaneously with primary antibodies to each of the tags, and IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image. b) His6-tagged GFAP proteins (amino acids 204—end) were expressed together with GST-RAP1 (lanes 1 through 4) or individually (lanes 5 through 8). Lysates (inputs) were mixed with glutathione beads, and eluted proteins were resolved on the gel. The blot was simultaneously probed with antibodies to GST and His6, and IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image. c) The GST and His6-tagged proteins were expressed together in HMS174(DE3) cells. Where indicated, a third protein expressed separately in HMS174(DE3) cells was added. Only eluates are shown for simplicity. The blot was simultaneously probed with antibodies to GST, His6, and MBP. IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image.