Fig 2: A STAT3 inhibitor reversed the ability of KPNA2 to promote angiogenesis in vitro. AAdenovirus-infected HUVEC overexpressed KPNA2 and were treated with a STAT3 inhibitor, and tube formation assays were carried out under hypoxia. Images were taken after 6 h. Scale bar: 200 µm. B Total tube length from tube formation assays. C Adenovirus-infected HUVEC overexpressed KPNA2 and were treated with a STAT3 inhibitor before Transwell assays performed under hypoxia. Images were taken after 6 h. Scale bar: 200 µm. D Statistical analysis of the results of Transwell assays (cells/fields). E Adenovirus-infected HUVEC overexpressing KPNA2 were treated with a STAT3 inhibitor and subjected to EdU/DAPI staining under hypoxia (red: EdU, blue: DAPI). Scale bar: 200 µm. F Statistical analysis of the EdU/DAPI staining results. G The binding of STAT3 and KPNA2 promotes the binding of STAT3 and JAK1 and phosphorylation of STAT3. P-STAT3 enters the nucleus, where it promotes the expression of VEGF and ANGPT2, which promotes angiogenesis. Each experiment was repeated three times. * p < 0.05

Fig 3: The binding of KPNA2 and STAT3 was increased under hypoxia. A IP-MS of KPNA2 showed that the binding of 364 proteins to KPNA2 was increased under hypoxia compared to normoxia. Metascape was used for enrichment analysis of these proteins, and the results showed that the top three enriched pathways were RNA metabolism, ribonucleoprotein complex biogenesis, and the VEGFA-VEGFR2 pathway. B After overexpression and knockdown of KPNA2 in HUVEC under hypoxia for 12 h, changes in VEGF and P-VEGFR2 levels were detected by Western blotting, and GAPDH was used as an internal control. C We selected the top ten proteins related to the VEGFA-VEGFR2 pathway upregulated under hypoxia (IgG as a control) and the top ten proteins related to the VEGFA-VEGFR2 pathway that were found to be upregulated under hypoxia vs. normoxia identified by IP-MS analysis of KPNA2 in HUVEC and determined the intersecting proteins. We obtained three proteins, namely, GPX1, STAT3 and MOV10. D The changes in MOV10 and GPX1 levels were detected by Western blotting after overexpression or knockdown of KPNA2 in HUVEC under hypoxia for 12 h, and ß-ACTIN was an internal control. E Western blotting analysis of immunoprecipitated STAT3 under normoxia vs hypoxia for 12 h was used to detect KPNA2. F IP analysis of KPNA2 under normoxia vs hypoxia for 12 h was carried out, and Western blotting was used to detect STAT3. Each experiment was repeated three times

Fig 4: 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 5: Overexpression of KPNA2 promoted angiogenesis in vitro and in vivo under hypoxia. A HUVEC were infected with Ad-KPNA2 to overexpress KPNA2. After 48 h of infection, tube formation assays were carried out under normoxia and under hypoxia, and pictures were taken 6 h later. Scale bar: 200 µm. The total lengths of the tube formation assay were analyzed statistically. Each experiment was repeated three times. B HUVEC were infected with Ad-KPNA2 to overexpress KPNA2. After 48 h of infection, Transwell assays were performed under hypoxia, and pictures were taken 6 h later. Scale bar: 200 µm. For each experiment, 3 fields of view were randomly selected for cell counting, and the average value was taken for statistical analysis. Each experiment was repeated three times. C HUVEC were infected with Ad-KPNA2 to overexpress KPNA2. After 48 h of infection, EdU staining was carried out under hypoxia, and pictures were taken 6 h later. Scale bar: 200 µm (EDU: red, DAPI: blue). EDU/DAPI were analyzed statistically. Each experiment was repeated three times. D Changes in blood perfusion in the ischemic hindlimb of model mice after overexpression of KPNA2 before and after surgery and at 3 days, 7 days, and 14 days. n = 5. E CD31 immunofluorescence (green) analysis of the gastrocnemius muscle of the ischemic mouse hindlimb at 14 days after KPNA2 overexpression. Scale bar 100 µm. n = 5. F Statistical analysis of blood flow changes in the ischemic hindlimb of model mice after overexpression of KPNA2 before and after surgery and at 3, 7, and 14 days. n = 5. G Vessel density statistics of CD31 immunofluorescence of the gastrocnemius muscle of the ischemic mouse hindlimb at 14 days after KPNA2 overexpression. n = 5. H KPNA2 expression of the gastrocnemius muscle of the ischemic mouse hindlimb at 14 days after KPNA2 overexpression detected by Western Blot. n = 5. * p < 0.05. NonHLI non-hindlimb ischemia, HLI hindlimb ischemia