Fig 2: VEGF165 induced activation of pERK/cyclinE is partly involved in the increased proliferation of HUVEC promoted by CREG. (A–D) VEGF165 expression was detected in H–S cells (A and B) or in HUVEC-AdCREG cells (C and D) by Western blotting, with ß-tubulin used as a loading control. Pooled analysis of VEGF165 levels were assessed after normalization to ß-tubulin with HUVEC as controls. *p < 0.05; **p < 0.01. Data are given as the mean ± SD (n = 3). (E–I) HUVEC-AdCREG cells were treated with neutralizing antibody of VEGF165 (5 µg/mL, 25 µg/mL and 50 µg/mL). FCM analysis (E and F), BrdU incorporation assay (G) and cell counting (H and I) were performed to assess the proliferation. Pooled analysis of (S + G2)%, the ratio of BrdU+ cells and the cell number were plotted, respectively. *p < 0.05; **p < 0.01; ***p < 0.01. Data are given as the mean ± SD (n = 3). (J–N) HUVEC-shCREG (H–S) cells were treated with recombinant human VEGF165 (5 ng/mL, 10 ng/mL or 20 ng/mL). FCM analysis (J and K), BrdU incorporation assay (L) and cell counting (M and N) were performed to assess the proliferation. Pooled analysis of (S + G2)%, the ratio of BrdU+ cells and the cell number were plotted, respectively. *p < 0.05; **p < 0.01; ***p < 0.01. Data are given as the mean ± SD (n = 3). (O–R) Levels of p-ERK, ERK, cyclin E and ß-tubulin were detected by Western blotting either in HUVEC-AdCREG cells treated with VEGF165 (50 µg/mL) neutralizing antibody (O and P) or in H–S cells pre-incubated with recombinant VEGF165 (20 ng/mL) (Q and R). Pooled quantitative analysis of p-ERK and ERK and cyclin E expression were plotted after normalization to ß-tubulin. Data are given as the mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.01.

Fig 3: Downregulation of CREG suppresses the proliferation of HUVEC. (A) Stable HUVEC clones with CREG silenced down (H–S) or expressing a scrambled negative control shRNA sequence (scramble) were established by retroviral infection and puromycin selection. Cell lysates were collected, and the expression of CREG was detected by Western blotting with ß-tubulin used as a loading control; (B) Pooled analysis of CREG protein levels assessed after normalization to CREG/ß-tubulin of the HUVEC group. Scramble HUVEC were used as a negative control. Data are given as the mean ± SD (n = 3). *p < 0.05; ***p < 0.001; NS: no significant difference; (C) Cell counting by hemocytometer. A total of 2 × 105 cells in the logarithmic phase in each of the three groups were plated in four 10-cm diameter dishes. After 24 h, cells were trypsinized and counted by hemocytometer. **p < 0.01 compared with the HUVEC and scramble group; (D) BrdU incorporation assay. Data are given as the mean ± SD (n = 3). **p < 0.01; ***p < 0.001; NS: no significant difference; (E) Effects of the downregulation of CREG on HUVEC cell cycle progression assessed by FCM; (F) Cell proliferation was accessed by the percentage of (S + G2) phase cells in the cell cycle. Data are given as the mean ± SD (n = 3). **p < 0.01 compared with the HUVEC and scramble group. NS: no significant difference.

Fig 4: ERK, but not PI3K/Akt, mediates the increase of cyclin E induced by CREG overexpression in HUVEC. (A) Expression of p-ERK, ERK, p-Akt, Akt, cyclin E and ß-tubulin was detected by Western blotting in the three groups of cells with LY294002 (50 µM) or PD098059 (20 µM) treatment. Vehicle (DMSO)-treated cells were used as control, and ß-tubulin was used as a loading control; (B,C) Pooled analysis of the levels of Akt (B) and ERK (C) signaling activation accessed by the grayscale ratios of p-Akt/Akt and p-ERK/ERK, respectively. Data are given as the mean ± SD (n = 3). *p < 0.05; **p < 0.01. NS: no significant difference. (D) After normalization to ß-tubulin, the difference in the expression of cyclin E between the HUVEC-AdGFP and HUVEC-AdCREG group was reduced by the treatment of PD098059, but not LY294002. Data are given as the mean ± SD (n = 3). **p < 0.01. NS: no significant difference.

Fig 5: CREG overexpression induces the expression of cyclin E in HUVEC in both protein and mRNA levels. (A) Effects of CREG overexpression on the protein expression of various cell cycle regulators. HUVEC and HUVEC-AdGFP were used as controls. The expression of different cyclins (B1, D1, D3 and E), CDKs (CDK2, CDK4, CDK6) and cell division cycle 2 (Cdc2) was detected by Western blotting with ß-tubulin as a loading control; (B,C) Pooled quantitative analysis of the protein expression of cyclins (B), CDKs and Cdc2 (C) against ß-tubulin. *p < 0.05. Data are given as the mean ± SD (n = 3). (D) The mRNA expression of cyclin E in different cell groups was detected by RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. M: molecular marker; (E) Pooled analysis of cyclin E mRNA levels quantified by grayscale analysis of the ratio of cyclin E/GAPDH with Quantity One software. Data are given as the mean ± SD (n = 3). *p < 0.05; **p < 0.01. NS: no significant difference.