Fusion fx7 system

The supernatant fluid of HUVECs culture was applied to a 10% polyacrylamide gel and electrophoresed at 60–90 V to investigate the components of SIRT7. The SIRT7 proteins were then transferred to nitrocellulose membranes. The membranes were blocked with nonfat dry milk for 2 h and incubated overnight with anti-SIRT7 antibodies (DF6161, Affinity Biosciences, Jiangsu, China). HRP-conjugated goat anti-rabbit IgG (BA1039, Boster Biological Technology, Hubei, China) was used as the secondary antibody. The membranes were briefly incubated with ECL detection reagent (ECL-00114, Dingguo, Beijing, China). The protein was visualized and analyzed using the Fusion FX7 system (Peqlab, Erlangen, Germany). Anti-GAPDH antibodies (AB-P-R001,Goodhere Biotechnology, Zhejiang, China) was used as an internal control. All raw Western blot data of this article are presented in Supplementary Figure S3.

Tumor cell lysates were applied to polyacrylamide gels and separated for 10 min at 80 V and for 60–90 min at 120 V. The proteins were then transferred to nitrocellulose membranes. After blocking with non-fat dry milk for 1 h, the membranes were incubated overnight with the following primary antibodies: CDK1 (mouse IgG1, clone 1, dilution 1:2500), pCDK1 (Mouse IgG2a, clone 55, dilution 1:2500), CDK2 (mouse IgG2a, clone 55, dilution 1:2500), Cyclin A (Mouse IgG1, clone 25, dilution 1:500), Cyclin B (Mouse IgG1, clone 18, dilution 1:1000), and p27 (Mouse IgG1, clone 57/Kip1, dilution 1:500) (all BD Biosciences) as well as pCDK2 (Rabbit, polyclonal antibody, dilution 1:1000) and p19 (Rabbit IgG, clone 12D1, dilution 1:1000) (both Cell Signaling). HRP-conjugated rabbit-anti-mouse IgG or goat-anti-rabbit IgG (IgG, both: dilution 1:1000, Dako, Glosturp, Denmark) served as secondary antibodies. The membranes were incubated with an ECL detection reagent (ECL; Amersham/GE Healthcare, München, Germany) and then analyzed by the Fusion FX7 system (Peqlab, Erlangen, Germany). β-Actin served as the internal control (1:1000; clone AC-15; Sigma-Aldrich, Taufenkirchen, Germany). The ratio of protein intensity/β-actin intensity was calculated for pixel density analysis of the protein bands and illustrated as percentage of controls (GIMP 2.8 software, www.gimp.org, accessed on 9 October 2021).

Western blot analysis concentrated on proteins involved in tumor cell differentiation and migration processes and included E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression. Tumor cell lysates were applied to a polyacrylamide gel and run for 90 min at 100 V. The concentration of acrylamide ranged from 3% to 12%, depending on the evaluated protein. The proteins were then transferred to nitrocellulose membranes for 1 h at 100 V and blocked with non-fat dry milk (1 h). Overnight incubation was finally carried out with monoclonal antibodies directed against E-cadherin, N-cadherin, vimentin, and CK 8/18 (all were from Cell Signaling, Leiden, The Netherlands). As secondary antibodies, HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were used at a 1:5000 dilution (both were from Upstate Biotechnology, Lake Placid, NY, USA). Proteins were visualized by the ECL detection reagent (Amersham/GE Healthcare, München, Germany) and analyzed with the Fusion FX7 system (Peqlab, Erlangen, Germany). Internal controls were carried out with an anti-β-actin antibody (clone AC-15; Sigma-Aldrich). To quantify the intensity of the protein bands, the protein/β-actin intensity ratio was quantified using GIMP 2.8 software.