Fig 2: KPNA2 promotes STAT3 phosphorylation and the nuclear accumulation of phosphorylated STAT3. A Western blot analysis of the changes in KPNA2, STAT3, P-STAT3 and ANPTG2 levels in HUVEC under hypoxia for 12 h after overexpression and knockdown of KPNA2. GAPDH was used as an internal reference. B KPNA2 was overexpressed and knocked down in HUVEC under hypoxia for 12 h, and P-STAT3 was detected by Western blotting after separation of the nuclear and cytoplasmic proteins. The nuclear reference was Laminb1, and the cytoplasmic reference was GAPDH. C Immunofluorescence detection of P-STAT3 (red) and DAPI staining of the nuclei (blue) under hypoxia for 12 h after KPNA2 was overexpressed or knocked down in adenovirus-infected HUVEC. Scale bar: 25 µm D Quantitative RT-PCR detection of VEGF and ANGPT2 mRNA expression under hypoxia in HUVEC in which KPNA2 was overexpressed or knocked down. E After adenovirus infection of HUVEC to knock down KPNA2 under hypoxia for 12 h, STAT3 was assessed by Co-IP, and Western blotting was used to detect JAK1, JAK2, SRC, TYK2, and STAT3. F KPNA2 was knocked down in HUVEC under hypoxia for 12 h, and P-JAK1 was detected by Western blotting. G Molecular simulations with ZDOCK showed possible interactions between S220 (KPNA2)-Y657 (STAT3), N228 (KPNA2)-Q643 (STAT3), and Y225 (KPNA2)-Q644 (STAT3). H Constructs from truncated plasmids corresponding to 4 domains of STAT3 with added Flag tags were prepared; the constructs corresponded to amino acids 1–137, 138–319, 320–493, and 494–770. I Four truncated STAT3 plasmids were transfected into 293 T cells, KPNA2 was used for Co-IP, and Flag was detected by Western blotting. Each experiment was repeated three times. NC negative control

Fig 3: ANGPT2 is required for HOXD9-mediated proliferation, cell cycle progress and apoptotic inhibition of NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and the plasmid expressing ANGPT2 (exANGPT2) or empty vector. NCI-H661 cells were co-transfected with exHOXD9 and siANGPT2 or siNC. A After 48 h of transfection, ANGPT2 mRNA expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by real time-PCR assay. B After 48 h of transfection, ANGPT2 protein expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by western blot assay. C After 48 h of transfection, NSCLC cells were seeded into 96-well microplates and analyzed with CCK-8 reagents at indicated time. D Cell cycle progression in two NSCLC cells was evaluated by flow cytometry analysis. E Apoptosis in NCI-H292 cells was evaluated by flow cytometry analysis. The apoptotic rate was quantified by adding the percentages of early apoptotic cells (Annexin V+/PI- in Q4 quadrant) and late apoptotic cells (Annexin V+/PI+ in Q2 quadrant). * p < 0.05; ** p < 0.01; *** p < 0.001. Data in (A), (C), (D) and (E) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials

Fig 4: ANGPT2 is involved in the anti-migratory and anti-invasive effects of HOXD9 silencing in NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and exANGPT2 or empty vector. NCI-H661 cells were co-transfected with exHOXD9 and siANGPT2 or siNC. (A and B) After 48 h of transfection, the migratory ability and invasiveness of NCI-H292 (A) and NCI-H661 (B) cells were evaluated by scratch-wound healing assay (Scale bar = 200 µm) and transwell assay (Scale bar = 100 µm). ** p < 0.01; *** p < 0.001. Data in (A) and (B) are presented as mean values ± SD

Fig 5: ANGPT2 blocks the suppression of PD-L1 expression by HOXD9 in NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and exANGPT2 or empty vector. A After 48 h of transfection, total PD-L1 protein expression in NCI-H292 cells was evaluated by western blot assay. B After 48 h of transfection, membrane PD-L1 protein expression in NCI-H292 cells was evaluated by flow cytometry analysis. MFI, relative mean fluorescence intensity. * p < 0.05. Data in (B) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials