br All animal experiments were conducted according to the in
All animal experiments were conducted according to the institu-tional ethics and safety guidelines (Institutional Animal Welfare and
Ethics Committee, First Affiliated Hospital of Bengbu medical college, Anhui, China). In total, forty 8-week-old BALB/c-Foxn1null mice were used in the experiments (Model Animal Research Centre of Nanjing University, Nanjing, China). The mice were randomly divided into four groups. APPBP2 silenced and control xenograft model sets from A549 (or H1299) were established using 5 × 106 L-Glutamine injected into the right flanks of the subcutaneous tissue of the mice. Tumour length, width and tumour weight were measured every three to four days until the experiment ended. The tumour volume was calculated based on the conventional formula: V = (L × W2)/2, where L and W are the biggest and smallest diameters of the tumour.
2.12. Coimmunoprecipitation (co-IP)
To conduct the experiments, four additional plasmids were con-structed. Flag, Flag-APPBP2, His, and His-PPM1D were cloned into pCDNA 3·1, respectively. For APPBP2 co-IP in A549 (or H1299) cells,
the cells transformed by lipo-3000, expressing Flag-APPBP2, PPM1D, and SPOP, were cultured for 72 h. Meanwhile, the transformed cells expressing Flag, APPBP2, PPM1D, and SPOP were set as the negative control group. For PPM1D co-IP in the A549 (or H1299) cells, the trans-formed cells expressing His-APPBP2, PPM1D, and SPOP were cultured for 72 h. The transformed cells expressing His, APPBP2, PPM1D, and SPOP were set as the control group. The cells were lysed for co-IP and were incubated with beads (pierce, 6149) which covalently pre-couple Flag antibody (abcam,ab205606) or His antibody (abcam, ab18184). Some cell lysis, which was incubated with the beads cova-lently pre-coupled with human IgG, was set as another negative control. The protein binding complex was isolated by centrifuging. The precipi-tates were diluted with SDS sample buffer, separated on a 10% SDS– PAGE gel and subjected to immunoblotting with the corresponding antibodies.
2.13. Reanalysing the TCGA database
The investigators explored the mRNA level of APPBP2 in The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) cohort. The RNA Seq V2 RSEM data of TCGA-LUAD was obtained using TCGAbiolinks (https://github.com/BioinformaticsFMRP/TCGAbiolinks,
R version 3.5). The investigators identified candidate genes which may correlate with the APPBP2 mRNA level using cBioPortal tool [12,13].
2.14. Statistical analysis
Data are presented as mean ± SEM and two-tailed t-test was used to determine statistical significance difference between two groups (ex-cept Supplementary Fig. s3b, which adopted one-tailed t-test). One-way ANOVA followed by a Tukey's Multiple Comparison test was applied for the comparison among groups in the same graph. A p-value of b0.05 was considered statistically significant.
3.1. The elevated expression of APPBP2 in human NSCLC
To explore the correlation of APPBP2 with NSCLC, we compared the microarray-based expression data of APPBP2 between the paired nor-mal and tumour tissues in two NSCLC cohorts from Spain (GEO acces-sion: GSE18842)  and Taiwan (GEO accession: GSE19804) . In both cohorts, APPBP2 was significantly up-regulated in the tumour tissues compared with the paired normal tissues (paired t-test: p b 0.0001 for the Spain cohort and p = 0·0013 for the Taiwan cohort) (Fig. 1a). In addition, primary cancers and the paired normal tumour-adjacent lung tissues were collected from ten NSCLC patients and subse-quently subjected to immunohistochemical and quantitative real-time PCR (qPCR)analysis. As expected, an elevated expression of APPBP2was observed in NSCLC tumours relative to normal lung tissues (Fig. 1b–e).
Fig. 1. The expression of APPBP2 was elevated in human NSCLC tissues. (a) The APPBP2 expression profile of the NSCLC tissues and the adjacent normal tissues from the GEO database. The left and right panels represent samples from Spain and Taiwan, respectively. (b,c) Immunostaining of APPBP2 of the NSCLC tumour tissues (lower) and the matched normal tissues (upper) from our hospital. Blue signals represent DAPI and brown signals represent APPBP2. (d) Image grey integral optical density (IOD) from immunohistochemistry results. Pink colour and blue colour represent normal tissues and NSCLC tissues, respectively. (e) The APPBP2 mRNA expression levels of ten paired LUAD patients were measured by RT-PCR. Pink colour and blue col-our represent normal tissues and LUAD tumour tissues, respectively. Data are presented as mean ± SEM. (Two-tailed t-test for group comparison) 0.001 b **p b 0.01, ***p b 0.001 for in-dicated comparison. Scale bar: 50 μm.