Fig 1: Exogenous sVASN could be internalized into different types of cells through cell surface CD71. (A) Western blot analysis of the internalization of exogenous sVASN into U251 or Ishikawa cells with siRNA treatment targeted to the CD71 gene. NC, negative control; si, siCD71; n = 3. (B) Western blot analysis of the internalization of exogenous sVASN in hCMEC/D3 or HaCaT cells with CD71 blockade treatment. n = 3. (C) Internalization of exogenous sVASN with CD71 blockade treatment visualized via LSCM. (D) Internalization of exogenous sVASN into the CD71 knockout cell line visualized via LSCM. The Western blot bands or the mean fluorescence intensity (MFI) were determined via ImageJ software. GAPDH was used as an internal control. All of the data are presented as the means ± SDs. All of the assays were conducted in triplicate. Statistical analysis in (A) was performed using two-way ANOVA followed by Sidak’s multiple comparisons test, in (B) using two-way ANOVA followed by Tukey’s multiple comparisons test, in (C) using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test and in (D) using an unpaired t test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant compared with the indicated groups.
Fig 2: sVASN inhibited T cell activation through cell surface CD71. (A) Western blot analysis of the mTOR-AKT signaling pathway after sVASN treatment in activated Jurkat cells. n = 3. (B) Western blot analysis of the mTOR-AKT signaling pathway in activated Jurkat cells treated with sVASN with or without VASN-specific monoclonal antibody (mAb) counteraction. n = 3. (C) Activated Jurkat cells were treated with increasing doses of sVASN or CD71 blockade, and the fold change in the level of IL-2 was measured. Actv., activated Jurkat cells. GAPDH was used as an internal control. All of the data are presented as the means ± SDs. All of the assays were conducted in triplicate. Statistical analyses in (A,B) were performed using two-way ANOVA followed by Tukey’s multiple comparisons test and in (C) using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant compared with the indicated groups.
Fig 3: Exogenous sVASN was conducive to the proliferation of cancer cells through cell surface CD71. (A) Proliferation assay of Ishikawa cells treated with exogenous sVASN. (B) The tumorigenesis of Ishikawa cells in vivo after treatment with exogenous sVASN, CD71 blockade, CD71 blockade followed by sVASN or a blank. The tumor volumes (left panel) were measured every three days, and the tumor weights (middle panel) and images (right panel) of the tumors from the nude mice at autopsy are presented. n = 5. (C,D) Western blot analysis of the YAP1/TAZ signaling pathway in Ishikawa cells treated with sVASN with or without the CD71-blocking peptide (C), or with the counteracting VASN-specific monoclonal antibody (mAb) (D). n = 3. The Western blot bands were determined via ImageJ software. GAPDH was used as an internal control. All of the data are presented as the means ± SDs. All of the assays were conducted in triplicate. Statistical analyses in (A) and (B, left) were performed using one-way ANOVA with Dunnett’s multiple comparison test, in (B, right) and (D) using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test and in (C) using two-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant compared with the indicated groups.
Fig 4: CD71 was identified as a sVASN-binding protein. (A) Co-IP analysis of the binding of sVASN to full-length CD71. The sVASN plasmid was constructed with a His tag, and the CD71 plasmid was constructed with a Flag tag, both of which are in the C-terminus. (B) BIAcore analysis of the binding of sVASN to CD71. CD71 was labeled with biotin at a concentration of 50 nM. (C) ELISA analysis of the binding of sVASN to CD71. CD71 was coated at a concentration of 10 µg/mL. The concentrations of sVASN used were 50, 40, 30, 20, 10, 5, 2.5 and 0 µg/mL. (D) Co-IP analysis of the binding of sVASN-dom1 to full-length CD71. sVASN-dom1, 1–240 aa of VASN with a His tag at the C-terminus. (E) Co-IP analysis of the binding of sVASN-Cdel to full-length CD71. sVASN-Cdel, the N-terminus of sVASN, is a 1–220 aa fragment of VASN with a His tag at the C-terminus. (F) Co-IP analysis of the binding of VASN-Vp1 to full-length CD71. sVASN-Vp1, 1–135 aa of VASN with a His tag at the C-terminus. (G) Saturation binding of sVASN to CD71. CD71 was coated at a concentration of 1 µg/mL. The binding substrate sVASN ranged from 200 µg/mL to 3.125 µg/mL. The equation for the one-site binding model: [Y = Bmax×X/(Kd + X)]. All of the assays were conducted in triplicate.
Fig 5: sVASN promoted the angiogenesis of endothelial cells through cell surface CD71. (A–C) Angiogenesis of brain microvascular endothelial cells (hCMECs/D3) treated with exogenous sVASN, CD71 blockade, CD71 blockade followed by sVASN or the blank. Representative images (A), statistics of branch points (B) and tube length (C) are shown. n = 6. (D) Western blot analysis of the VEGF signaling pathway in hCMEC/D3 cells treated with or without CD71 blockade. n = 3. (E) Western blot analysis of the VEGF signaling pathway in hCMEC/D3 cells treated with sVASN with or without a VASN-specific monoclonal antibody (mAb) counteraction. n = 3. The Western blot bands were determined via ImageJ software. GAPDH was used as an internal control. All of the data are presented as the means ± SDs. All of the assays were conducted in triplicate. Statistical analyses in (B,C) were performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test and in (D,E) using two-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant compared with the indicated groups.
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