Fig 2: SCG2 protein inhibits CRC tumor growth in mice. (A) IgG and SCG2 (10 µg/mice/time) were injected into the tumors at 8, 10 and 12 days post-SW620 cell injection. (B) Image of SW620 tumors treated with IgG or SCG2 protein. (C) At 8 days post-injection, the SW620 tumor length and width were measured every 3 days and a tumor growth curve was generated (n = 5, **P < 0.01, mean ± SD, t-test). (D) Analysis of SW620-IgG and SW620-SCG2 tumor weights (n = 5, **P < 0.01, mean ± SD, t-test). IHC staining of SCG2 (E), HIF-1a (F), and VEGF (G) expression in SW620-IgG and SW620-SCG2 tumors (n = 4). Scale bar: 100 µm. Analysis of SCG2-, HIF-1a- and VEGF-positive cells in SW620 tumors (n = 4, **P < 0.01, mean ± SD, t-test).

Fig 3: SCG2 inhibits angiogenesis via downregulating VEGF expression. (A) Sequencing of coding RNA in SW620-Ctrl and SW620-SCG2 tumors. (B) KEGG analysis of deregulated coding RNA between SW620-Ctrl and SW620-SCG2 tumors. (C) qPCR analysis of VEGF mRNA expression in the indicated tumors (n = 3, **P < 0.01, mean ± SD, t-test). (D) IHC staining of VEGF expression in SW620-Ctrl and SW620-SCG2 tumors. Scale bar: 100 µm. Analysis of VEGF-positive cells in SW620 tumors (n = 4, **P < 0.01, mean ± SD, t-test). (E) qPCR analysis of VEGF mRNA expression in the indicated cells (n = 3, **P < 0.01, mean ± SD, t-test). (F) Proteins of the indicated cells were extracted for western blot analysis. (G) The cell culture supernatants of indicated cells were collected for use in an ELISA (n = 3, **P < 0.01, mean ± SD, t-test). (H, I) VEGF protein was added to the cell culture supernatant of SW620-SCG2 cells to rescue VEGF expression. The conditioned medium was used for the HUVEC tube formation assay (H) and invasion assay (I). Scale bar: 100 µm (n = 3, **P < 0.01; ns, no significant difference, mean ± SD, t-test).

Fig 4: SCG2 impairs CRC tumor growth. (A) Proteins were extracted from CRC cells and HIEC for western blot analysis. (B) SW620 and RKO cells were stably infected with lenti-control (Ctrl) and an SCG2 overexpression system. Proteins were extracted from SW620-Ctrl, SW620-SCG2, RKO-Ctrl, and RKO-SCG2 cells for western blot analysis. (C) IHC staining of SCG2 expression in SW620-Ctrl and SW620-SCG2 tumors. Scale bar: 100 µm. Analysis of SCG2-positive cells in SW620 tumors (n = 4, **P < 0.01, mean ± SD, t-test). (D) Images of SW620-Ctrl and SW620-SCG2 tumors. (E) By 10 days postinjection, the SW620-Ctrl and SW620-SCG2 tumor length and width were measured every 3 days and a tumor growth curve was generated (n = 5, **P < 0.01, mean ± SD, t-test). (F) Analysis of SW620-Ctrl and SW620-SCG2 tumor weights (n = 5, **P < 0.01, mean ± SD, t-test). (G) Images of RKO-Ctrl and RKO-SCG2 tumors. (H) By 10 days postinjection, the RKO-Ctrl and RKO-SCG2 tumor length and width were measured every 3 days and a tumor growth curve was generated (n = 5, **P < 0.01, mean ± SD, t-test). (I) Analysis of RKO-Ctrl and RKO-SCG2 tumor weights (n = 5, **P < 0.01, mean ± SD, t-test).

Fig 5: SCG2 promotes HIF-1a degradation via binding to VHL. (A, B) Proteins of the indicated cells were extracted for western blot analysis. (C) The indicated cells were treated with MG132. After 4 h, proteins were extracted for western blot analysis. (D) Proteins of SW620-SCG2 cells were extracted for co-immunoprecipitation analysis. The immunoprecipitated proteins were used for western blot analysis. (E) IHC staining of HIF-1a expression in SW620-Ctrl and SW620-SCG2 tumors. Scale bar: 100 µm. Analysis of HIF-1a-positive cells in SW620 tumors (n = 4, **P < 0.01, mean ± SD, t-test). (F, G) The indicated cells were treated with DMOG. After 24 h, proteins were extracted for western blot analysis (F) and the cell culture supernatant was collected for use in an ELISA (G, n = 3, **P < 0.01, mean ± SD, t-test).