PRMT5 facilitates angiogenesis and EMT via HIF-1α/VEGFR/Akt signaling axis in lung cancer

PRMT5 is highly expressed in human lung cancer

To define the biological functions of PRMT5 in lung cancer, we first evaluated the relative expression levels of PRMT5 in lung tumor tissues and normal tissues. As seen in Figure 1A, PRMT5 mRNA expression was much higher in lung tumor tissues than in normal tissues, indicating that PRMT5 is up-regulated in lung cancer. Next, we assessed the association of PRMT5 expression with the lung tumor stages. As seen in Figure 2B, PRMT5 expression was much higher in stage II, stage III, and stage IV than in stage I, and the ectopic expression levels of PRMT5 were positively correlated with the stages. Furthermore, the patients with high expression levels of PRMT5 had a low survival rate compared with the patients with low expression levels of PRMT5 (Figure 1C). To validate the above findings, we collected the eight paired-lung tumor tissues and normal tissues from the patients with lung cancer and the PRMT5 protein expression were determined by Western blotting. As seen in Figure 1D and 1E, PRMT5 protein expression levels were significantly elevated (4.4 fold) in tumor samples compared with normal samples. Additionally, we detected the PRMT5 protein expression in lung cancer cell lines and normal human fetal lung fibroblast cells (IMR90). Consistent with the above lung cancer tissue results, PRMT5 was overexpressed in lung cancer cells than IMR90 cells (Figure 1F, 1G). Previous studies showed that HIF-1α and PI3K/Akt signaling pathway was closely related to angiogenesis, metastasis, and EMT [20, 21]. We next assessed the correlation between PRMT5 and HIF-1α or PI3K/Akt signaling pathway. As seen in Figure 1H, 1I, we found that PRMT5 positively correlated with HIF-1α and PI3K/Akt signaling pathway in lung cancer, suggesting a potential association between these genes and pathways. Taken together, our findings indicate that abnormal expression of PRMT5 is closely related to the lung tumor stages and survival rates in patients with lung cancer, and PRMT5 may regulate angiogenesis, metastasis, and EMT through HIF-1α and PI3K/Akt signaling pathway.

Figure 1. Overexpression of PRMT5 results in human lung cancer progression. (A) PRMT5 mRNA expression level was analyzed in adjacent normal tissues (n=486) and lung tumor tissues (n=338) using TCGA database. ****P <0.0001 vs. normal tissues with Mann-Whitney test. (B) PRMT5 was closely associated with stages in human lung cancer with analysis of the TCGA database. N=113 for stage I; n=68 for stage II; n=43 for stage III and n=19 for stage IV. P <0.001 vs. indicated stages. (C) PRMT5 expression level was negatively correlated with the patient’s overall survival with TCGA database analysis. n=239 for PRMT5-low group and PRMT5-high group. P values were determined by log-rank test and p=0.048. (D) PRMT5 protein expression levels were assessed by Western blotting in adjacent normal tissues (N) and lung tumor tissues (T). (E) PRMT5 protein expression levels were quantified in adjacent normal tissues and lung tumor tissues (n=8, each group). *P < 0.05 vs. normal tissue. (F) PRMT5 protein expression was detected by Western blotting in the indicated cell lines. (G) PRMT5 protein expression levels were quantified in the indicated cell lines. (n=3). *P < 0.05 vs. IMR90 cells. (H, I) The correlations between PRMT5 and HIF-1α or PI3K/Akt pathway were analyzed. The correlations between individual gene and pathway score were analyzed with Spearman method as well. The abscissa represents the distribution of the gene expression, and the ordinate represents the distribution of the pathway score. The density curve on the right represents the trend in distribution of pathway immune score; the upper density curve represents the trend in distribution of the gene expression. The value on the top represents the correlation p value, correlation coefficient and correlation calculation method.

Figure 2. PRMT5 is induced by hypoxia. The HUVECs (A, B) and ASTC-a-1 cells (C, D) were treated with CoCl₂ (200μM) for indicated time points, and the mRNA expression levels of PRMT5 and HIF-1α were measured by qRT-PCR. *P < 0.05 vs. 0-time point, n=4. The HUVECs (E) and ASTC-a-1 cells (F) were treated with or without CoCl₂ (200μM) for 12h, and the HIF-1α expression induced by CoCl₂ was detected by immunofluorescence staining. Representative pictures were shown. Green= HIF-1α; Red=β-actin; Blue=DAPI. Scale Bar=50μm. The ASTC-a-1 cells (G) and HUVECs (H) were treated with CoCl₂ (200μM) for indicated time points, and the protein expression levels of PRMT5 and HIF-α were evaluated by Western blotting. (I) PRMT5 and HIF-α protein expression levels were quantified in ASTC-a-1 and HUVECs cells (n=3). *P < 0.05 vs. 0-time point. (J) The HUVECs were treated with VEGF (50ng/ml) in the presence or absence of GSK591, and the endothelial NO was monitored by the DAF-FM DA probe. Scale bar=50μm. (K) Quantitation of corresponding MFI values (n=6, each group). *P < 0.05 vs. control; ^#P < 0.05 vs. VEGF. (L) NOS enzymatic activity in HUVECs upon treatment of VEGF with or without GSK591 as evaluated by the Griess method. *P < 0.05 vs. control; ^#P < 0.05 vs. VEGF (50ng/ml) treatment. (n=3, each group). (M) Total NO was measured by ELISA kit using the Griess reaction in the supernatant of HUVECs upon treatment of VEGF (50ng/ml) with or without GSK591. *P < 0.05 vs. control; ^#P < 0.05 vs. VEGF treatment. (n=3, each group).

PRMT5 is induced by hypoxia, and NO production is suppressed by PRMT5 inhibition

PRMT5 is considered as an oncogene and overexpressed in many human cancers, but the role of PRMT5 in angiogenesis is still unclear. To dissect the function of PRMT5 in angiogenesis during lung cancer development, human umbilical vein ECs (HUVECs) were used in our study because HUVECs are a widely used cell model for studying vasculature and angiogenesis, and HUVECs also express many essential signaling molecules and endothelial markers associated with angiogenesis [22, 23]. We first determined the mRNA expression level of PRMT5 in HUVECs induced by CoCl₂ that mimics hypoxia in different in vitro studies. As seen in Figure 2A, PRMT5 mRNA expression was dramatically increased, induced by CoCl₂ in a time-dependent manner. Moreover, the HIF-1α, a marker for angiogenesis under hypoxia, was also markedly increased (Figure 2B). Hypoxia is one of the hallmarks of human lung cancer [24–26]. We wonder if the expression of PRMT5 and HIF-1α was changed in lung cancer cells induced by CoCl₂. To address this question, the ASTC-a-1 cells, a lung adenocarcinoma cell line, were used. As seen in Figure 2C, 2D, both PRMT5 and HIF-1α mRNA levels were significantly elevated in ASTC-a-1 cells in a time-dependent manner upon CoCl₂ treatment. In addition, we found that HIF-1α was highly expressed in the nucleus in both HUVECs (Figure 2E) and ASTC-a-1 cells (Figure 2F) in response to CoCl₂ treatment. Next, PRMT5 and HIF-1α protein expression was determined in HUVECs and ASTC-a-1 cells induced by CoCl₂. As seen in Figure 2G–2I, we found that PRMT5 and HIF-1α protein expression was increased in both HUVECs and ASTC-a-1 cells in a time-dependent manner.

NO plays an essential role in angiogenesis [27]. Thus, to determine whether PRMT5 is involved in NO production, the HUVECs were treated with VEGF in the presence or absence of GSK591, a potent PRMT5 inhibitor, and the NO production was monitored by the DAF-FM DA probe in living cells. As seen in Figure 2J, 2K, we found that NO production was strongly induced by VEGF compared with the control group, whereas NO production was markedly diminished by PRMT5 inhibitor GSK591. To further confirm whether PRMT5 is involved in NO production, the eNOS activity and total NO production in the medium of HUVECs were measured. As seen in Figure 2L, 2M, we found that both eNOS activity and total NO production were strongly induced by VEGF compared with the control group, whereas both eNOS activity and total NO production were lessened by PRMT5 inhibitor GSK591. Collectively, these results suggest that PRMT5 is engaged in NO production and angiogenesis, which may lead to EMT and metastasis during lung cancer progression.

Blocking PRMT5 impairs VEGFR2/Akt/eNOS signaling axis

VEGF/VEGFR2 signaling cascades, one of the most prominent angiogenetic factors, serve as an essential mediator to facilitate endothelial cell proliferation, survival, and migration, which promote abnormal angiogenesis. In order to dissect whether PRMT5 regulates angiogenesis and the possible molecular mechanism, we used the potent PRMT5 inhibitor to repress the PRMT5 enzyme activity. For this purpose, the HUVECs were treated with GSK591 for five days and then were stimulated with VEGF (50ng/ml) at indicated time points. As seen in Figure 3A, GSK591 treatment dramatically reduced the phosphorylation of VEGFR2 at both Tyr-1175 and Tyr966 induced by VEGF, and the symmetric dimethylation of arginine (SDMA) that was catalyzed by PRMT5 on a wide variety of targets was also reduced. In contrast, the total VEGFR2 was unchanged, indicating that blocking PRMT5 enzyme activity only affects the phospho-VEGFR2, but not the total protein. Furthermore, blocking PRMT5 also impaired the phosphorylation of Akt at both Thr-308 and Ser-473 and the phosphorylation of eNOS at Ser-1177 induced by VEGF, which were mainly engaged in endothelial cell duplication and angiogenesis. To further confirm whether PRMT5 regulates VEGFR2/Akt/eNOS signaling axis, we used lentivirus containing PRMT5-shRNA to knock down endogenous PRMT5, and the phosphorylation levels of VEGFR2, Akt, and eNOS were detected by Western blotting. As seen in Figure 3B, we found that down-regulation of PRMT5 attenuated the phosphorylation levels of VEGFR2, Akt, and eNOS induced by VEGF, similar to the GSK591 treatment. Altogether, these data indicate that PRMT5 indeed regulates angiogenesis via VEGFR2/Akt/eNOS signaling axis, which is related to PRMT5 enzyme activity.

Figure 3. Inhibiting PRMT5 impairs VEGFR2/Akt/eNOS signaling axis. (A) The HUVECs were incubated with vehicle or GSK591 (10μM) for five days, and the cells were serum starved and treated with VEGF (50ng/mL) at different time points. The cells were harvested, and the indicated protein expression levels were assessed by Western blotting (n=3). GAPDH served as an internal control. (B) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2, and the cells were serum starved. The cells were stimulated with VEGF (50 ng/mL), and the indicated protein expression levels were assessed by Western blotting (n=3). The PRMT5 activity was determined by the expression of SDMA.

Inhibiting PRMT5 attenuates HIF-1α expression and stability induced by hypoxia

HIF-1α is a heterodimeric transcription factor that plays a critical role in angiogenesis in response to ischemia or hypoxia [28]. To explore the potential function of HIF-1α and PRMT5 in lung cancer, we first evaluated the mRNA expression of HIF-1α in PRMT5 depletion cells. As seen in Figure 4A, down-regulation of PRMT5 did not affect the mRNA expression of HIF-1α, suggesting that a posttranslational mechanism may be involved in regulating HIF-1α protein levels by PRMT5. We next investigated whether down-regulation or inhibition of PRMT5 affects the protein expression of HIF-1α upon hypoxia stimulation. As seen in Figure 4B, 4C, both down-regulation and inhibition of PRMT5 strongly reduced the HIF-1α expression induced by CoCl₂. Moreover, the reduction of HIF-1α expression was negatively associated with the concentration of GSK591 (Figure 4D). These findings imply that PRMT5 may control HIF-1α expression via a posttranslational mechanism. To further explore how PRMT5 regulates HIF-1α expression and the related molecular mechanism, we detected the HIF-1α expression stability in PRMT5 depletion HUVECs or treatment of GSK591 in the presence of cycloheximide (CHX). As seen in Figure 4E, 4F, down-regulation or inhibition of PRMT5 markedly decreased the stability of HIF-1α protein in response to CoCl₂ and promoted its degradation. In order to understand which pathway mediated the degradation of HIF-1α, we used the bafilomycin A1 (BAF-A1), the lysosome inhibitor, and MG132, the proteasome inhibitor, to evaluate the HIF-1α degradation upon GSK591 treatment or in PRMT5 depletion HUVECs. As seen in Figure 4G, we found that HIF-1α degradation was entirely blocked by MG132 but not BAF-A1, indicating that PRMT5 regulated HIF-1α stability via the proteasome pathway, but not lysosome pathway. Finally, we wonder whether the reintroduction of PRMT5 could rescue the HIF-1α degradation in PRMT5 depletion HUVECs. To this end, we reintroduced WT-PRMT5 into the PRMT5 depletion HUVECs, and the cells were treated with CoCl_2. As seen in Figure 4H, the reintroduction of WT-PRMT5 rescued the HIF-1α expression in PRMT5 depletion HUVECs compared with vector control induced by CoCl₂. Collectively, our findings uncover a new mechanism by which PRMT5 promotes angiogenesis by regulating HIF-1α stability.

Figure 4. Suppressing PRMT5 reduces the expression and stability of HIF-1α induced by hypoxia. (A) The mRNA expression of HIF-1α and PRMT5 was measured by qRT-PCR in PRMT5 depletion cells (HUVECs, n=4). *P < 0.05 vs. Scr. N.S. means not significant. (B) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2, followed by CoCl₂ (200μM) treatment for 24h. The PRMT5 and HIF-α expression levels were determined by Western blotting (n=3). (C) The HUVECs were treated with vehicle or GSK591 (1μM) for five days and then were treated with CoCl₂ (200μM) for 24h. The HIF-1α expression level was determined by Western blotting (n=3). (D) The effect of different doses of GSK591 on the HIF-1α expression level induced by CoCl₂ as evaluated by Western blotting (n=3). (E) The HIF-1α stability was detected by Western blotting in PRMT5 depletion HUVECs upon cycloheximide (CHX, 20μg/mL) treatment at the indicated time points (n=3). (F) The HIF-1α stability was detected by Western blotting in GSK591-treated HUVECs upon cycloheximide treatment at the indicated time points (n=3). (G) The HUVECs were incubated with or without GSK591 (10μM) or infected with lentivirus containing PRMT5-shRNA2 and then pretreated with MG132 (10μM) and BAF-A1 (100nM) for 30 min before treating CoCl₂. The HIF-1α expression levels were evaluated by Western blotting (n=3). (H) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2 and then were transfected with vector or Flag-PRMT5 before treating CoCl₂. The HIF-1α expression levels were evaluated by Western blotting (n=3).

Inhibiting PRMT5 attenuates EMT and metastasis in lung cancer

Angiogenesis is a critical component of metastatic signaling and EMT by recruiting new blood vessels, which provides the principal route to facilitate the tumor cells to secede from the primary tumor site and enter circulation [29]. To probe the molecular basis of EMT and metastasis in lung cancer cells mediated by PRMT5, the ASTC-a-1 cells were treated with PRMT5 inhibitor GSK591 or infected with lentivirus expressing PRMT5-shRNA, and the EMT markers were monitored by immunofluorescence. As seen in Figure 5A–5D, inhibition of PRMT5 significantly repressed the expression of β-catenin, Vimentin, and Slug, whereas the expression of E-cadherin was increased upon GSK591 treatment. Similar results were obtained in PRMT5 depletion cells (Figure 5E–5H). To confirm the above findings, these markers were also detected by Western blotting. As seen in Figure 5I and 5J, the expression levels of β-catenin, Vimentin, and Slug were diminished in response to PRMT5-specific inhibitors GSK591 and EPZ or down-regulation of PRMT5. The PRMT5 enzyme activity was also reduced, indicated by the expression of SDMA (Figure 5K). Altogether, our findings suggest that PRMT5 regulates lung cancer cell EMT and metastasis, at least in part, via promoting angiogenesis by controlling HIF-1α/VEGFR/Akt/eNOS signaling pathway.

Figure 5. Blocking PRMT5 attenuates lung cancer invasion and metastasis. (A–D) The ASTC-a-1 cells were treated with GSK591 or Vehicle, and the EMT markers (β-catenin, Vimentin, Slug, and E-cadherin) were determined by immunofluorescence. Representative pictures were shown (n=3, each group). Scale Bar=50μm. (E–H) The ASTC-a-1 cells were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA1, and the EMT markers (β-catenin, Vimentin, Slug, and E-cadherin) were determined by immunofluorescence. Representative pictures were shown (n=3, each group). Scale Bar=50μm. (I) The ASTC-a-1 cells were infected with lentivirus containing scramble-shRNA, PRMT5-shRNA1, or PRMT5-shRNA2. The expression levels of EMT markers (β-catenin, Vimentin, and Slug) and PRMT5 knockdown efficiency were detected by Western blotting (n=3). (J) The ASTC-a-1 cells were treated with GSK591, and the expression levels of EMT markers (β-catenin, Vimentin, and Slug) were detected by Western blotting (n=3). (K) The ASTC-a-1 cells were treated with GSK591 or Vehicle or infected with lentivirus containing scramble-shRNA, PRMT5-shRNA1, or PRMT5-shRNA2. The PRMT5 enzyme activity was detected by Western blotting (n=3) using the SDMA antibody.

Clinical materials collection

The matched adjacent normal tissues and related lung tumor tissues were collected between January 2020 and January 2022 from patients who were accepted with surgical treatment at Minhang Hospital of Fudan University. A total of eight matched tissue samples were collected and were immediately flash-frozen in liquid nitrogen. Then, those samples were used for further experiments and assays.

Bioinformatics analysis

Bioinformatics analysis was performed with public datasets in TCGA and described previously [46]. We acquired TCGA datasets from Xena Browser. Gene expression dataset across normal and cancer tissue was extracted and analyzed. RNA expression values were transformed using the ‘voom’ method limma package, version 3.30.13. All multiple comparisons for TCGA analysis were corrected using Benjamini-Hochberg unless otherwise stated. For a direct comparison of the expression of PRMT5 between normal tissue and cancer tissues, an unpaired t-test or Mann-Whitney test was used, and p-values were calculated. TMM normalized counts were Log2 + 1 transformed and were used to classify patients into high and low expression groups for each gene of interest using the surv_cutpoint function in survminer R; then, four stages were generated from these data. The prognosis of each group was examined using Kaplan-Meier survival estimators with the survminer R package, version 0.4.1, with survival outcomes compared by log-rank tests. Moreover, RNA-sequencing expression (level 3) profiles and corresponding clinical information for PRMT5, HIF-1α and PI3K/Akt were downloaded from the TCGA dataset (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). R software GSVA package was used to analyze, choosing parameter as method=‘ssgsea’. The correlation between genes and pathway scores was analyzed by Spearman correlation. All the analysis methods and R packages were implemented by R version 4.0.3. p-value <0.05 was considered statistically significant.

Cell culture and inhibitors

The ASTC-a-1 cells were obtained from the Department of Medicine, Jinan University (Guangzhou, China), and the cells were cultured in Dulbecco’s Modified Eagle’s Medium (1:1) (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma cat# F2442), 50 units/ml penicillin, and 50 mg/ml streptomycin. The Human umbilical vein endothelial cells (HUVECs) were purchased from Thermo Fisher Scientific (cat# C0035C), and the cells were cultured in Endothelial Cell Growth Medium (EGM-2 BulletKit, Lonza, cat# CC-3162) supplemented with 10% FBS, L-glutamine, and 1% antibiotic solution. All other cell lines were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CAS) in Shanghai and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. The mycoplasma was measured by PlasmoTest™-Mycoplasma Detection Kit (InvivoGen, China) in ASTC-a-1cells and HUVECs before the experiments were performed. All the cells were maintained at 37° C with 5% CO₂ in an incubator. The ASTC-a-1 cells and HUVECs were treated with 200μM Cobalt Chloride (CoCl₂) for indicated time points to generate the hypoxic cell model. PRMT5 specific inhibitor EPZ015666 (GSK3235025, cat# HY-12727) and MG-132 (HY-13259) was purchased from MedChemExpress (MCE). The cycloheximide (cat# C4859) and GSK591 were purchased from Sigma (cat# SML-1751). The lysosome inhibitor bafilomycin A1 (BAF-A1, cat# 1334) was purchased from TOCRIS.

Construction of plasmids

The Scramble-shRNA and PRMT5-shRNAs were described previously [30]. Briefly, to knock down endogenous PRMT5, the Scramble-shRNA and PRMT5-shRNAs were cloned into a lentiviral vector. The human PRMT5 targeting sequences were: shRNA1, 5’-GGATAAAGCTGTATGCTGT-3’; shRNA2: 5’-GCCATCTATAAATGTCTGCTA-3’. To generate the lentivirus containing Scramble-shRNA or PRMT5-shRNAs, the helper plasmids MD2G and PAX2 were used (Addgene). The human PRMT5 cDNA was subcloned into the Flag vector and verified by sequencing.

Generation of PRMT5 stable knockdown cell line

The lentiviral constructs containing scramble-shRNA or PRMT5-shRNAs were co-transfected along with the helper plasmids MD2G and PAX2 into 293T cells using Lipofectamine™ 3000 (cat# L3000015, Invitrogen) transfection reagent according to the manufacturer’s protocol. After 24 h post-transfection, the culture media were replaced with fresh media, and then the culture media were harvested after 48 h post-transfection. The media were filtered, and the viral titer was pre-determined. Subsequently, ASTC-a-1 cells and HUVECs were infected with the lentivirus containing scramble-shRNA or PRMT5-shRNAs with an equal amount of virus particles. After 24 h, post-infection, the cells were selected with puromycin (Sigma, cat# p9620) for 48 h.

Gene expression analysis

Gene expression was performed by quantitative real-time PCR (qRT-PCR). The total RNA was extracted from ASTC-a-1 cells and HUVECs upon treatments using TRIzol reagent (Invitrogen, cat# 15596-018) according to the manufacturer’s protocol. After extraction of the total RNA, the concentration of the RNA was measured by NanoDrop 2000 spectrophotometers, and the equal amount of RNA (1ug) was applied to carry out the reverse transcription by C1000 Touch PCR Thermal Cycler (Bio-Red). Finally, the qRT-PCR was performed to detect the gene expression with SYBR green fluorescent Dye (Bio-Rad, cat# 1725272) by an ABI7500 PCR machine (Applied Biosystems™). The following primers were used in this study: human PRMT5 forward: 5’-CCTGTGGAGGTGAACACAGT-3’ and revise: 5’-AGAGGATGGGAAACCATGAG-3’; human HIF-1α, forward: 5′-CACCTCTTTTGGCAAGCATCCTG-3′ and revise: 5′-TATGAGCCAGAAGAACTTTTAGGC-3′; human GAPDH, forward: 5′-TGTGGGCATCAATGGATTTGG-3′ and revise: 5′-ACACCATGTATTCCGGGTCAAT-3. GAPGH served as an internal control. The relative mRNA expression level was calculated by the method of ΔΔ-Ct.

Western blot analysis

Western blot analysis was performed as described previously [47]. Briefly, the total proteins were extracted from tissues and cells with the lysis buffer (20 mmol/L Tris, PH 7.4, two mmol/L EDTA, two mmol/L EGTA, one mmol/L sodium orthovanadate, 1% Triton X-100, 150 mmol/L NaCl, 50 mmol/L sodium fluoride, 0.1% SDS, and 100 mmol/L phenylmethylsulfonyl fluoride) and then were centrifuged for 10 min at 4° C with the top speed of the centrifuge. Next, the Bradford method determined the protein concentration before separating the proteins in sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The proteins were transferred to the PVDF membranes (cat#1620177; Bio-Rad), and the membranes were washed three times with TBST and blocked using 5% non-fat milk for one h at room temperature. Next, the membranes were incubated with the indicated antibodies for overnight at 4° C: PRMT5 (Santa Cruz Biotechnology, cat# sc-376937), HIF-1α (Cell Signaling Technology, cat# 36169), Phospho-Tyr1175-VEGF Receptor 2 (Cell Signaling Technology, cat# 3770), phospho-Tyr996-VEGF Receptor 2 (Cell Signaling Technology, cat# 2474), total VEGFR 2 (Cell Signaling Technology, cat# 9698), phospho-Thr308-Akt (Cell Signaling Technology, cat# 4056), phospho-Ser473-Akt (cat# 4060; Cell Signaling Technology), total Akt (Cell Signaling Technology, cat# 4691), phospho-Ser1177-eNOS (Cell Signaling Technology, cat# 9570), total eNOS (Cell Signaling Technology, cat# 32027), Symmetric Di-Methyl Arginine Motif [sdme-RG] MultiMab™ (cat# 13222; Cell Signaling Technology), β-catenin (Cell Signaling Technology, cat# 8814), vimentin (Cell Signaling Technology, cat# 5741), Slug (Cell Signaling Technology, cat# 9585), Flag (Sigma, cat# F7425), β-actin (Santa Cruz Biotechnology, cat# sc-47778), and GAPDH (Cell Signaling Technology, cat# 5174). Then, the membranes were incubated with goat anti-rabbit conjugated to HRP secondary antibody (Santa Cruz Biotechnology, cat# sc-2004) or goat anti-mouse conjugated to HRP secondary antibody (Santa Cruz Biotechnology, cat# sc-2005) at room temperature for 2h. Finally, the proteins were detected by SuperSignal West Pico Chemiluminescent Substrate Western blotting reagents (Thermo Fisher Scientific, cat# 34580).

Immunofluorescence

ASTC-a-1 cells were seeded into 6-well plates for 24h before treatments. The cells were fixed for 20 min at room temperature, and then permeabilization was applied with ice-cold methanol at -20° C for 10 minutes. The cells were washed with PBS twice after permeabilization and incubated in blocking buffer (5% normal goat serum) for 60 min at room temperature, followed by incubation with the primary antibodies: HIF-1α (Cell Signaling Technology, cat# 36169), β-catenin (Cell Signaling Technology, cat# 8814), vimentin (Cell Signaling Technology, cat# 5741), Slug (Cell Signaling Technology, cat# 9585), E-Cadherin (Cell Signaling Technology, cat# 3195) and β-actin (Santa Cruz Biotechnology, cat# sc-47778) (diluted 1:50 in blocking buffer) at 4° C overnight. After incubation, the cells were washed with PBS three times, followed by incubation with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Thermo Fisher, cat# A-11005) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Thermo Fisher, cat# A-11034). Finally, the nuclei were labeled with DAPI (Sigma, cat# D9542) before observation. The images were analyzed with a confocal microscopy system (LSM700, Zeiss).

Nitric oxide synthase (NOS) activity measurement

To measure the NOS activity, HUVECs were treated with VEGF, GSK591, or vehicle, and the NOS activity was detected with the NOS activity assay kit (Abcam, ab211083) according to the manufacturer’s protocol. The NO synthesis activity was measured immediately once the samples were ready to use, and the substrate with cofactors was added immediately. The reactions were observed with Griess Reagents 1 and 2. The relative enzymatic activity of NOS was calculated with the absorbance at 540 nm.

Nitric oxides (NO) measurement

To measure the total nitric oxides (NO), the HUAECs were incubated with VEGF, GSK591, or vehicle, and the supernatants were collected, followed by deproteinizing with a ten kDa column spin cut-off system (SARTORIUS, cat# VS0101). Then, the assay was performed immediately by the NO assay kit (Abcam, ab65328) with the Griess method according to the manufacturer’s protocol. To detect the NO production in living cells, the dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; Invitrogen, cat# D23844) was used. The HUVECs were pretreated with DAF-FM DA for 15 min under indicated conditions, and the cells were washed for 10 min. The images were analyzed with a confocal microscopy system (LSM700, Zeiss), and the mean fluorescence intensity (MFI) was analyzed.

Statistical analysis

All experiments were performed in triplicate under identical conditions, and the data were shown as means ± SEM. Unpaired two-tailed Student’s t-test analyzed differences between the two groups. The difference with P < 0.05 was considered statistically significant.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.