Figure 2. RIOK1 promotes growth and metastasis of CRC cells in vitro and in vivo.

(A) The expression levels of RIOK1 in 7 CRC cell lines and normal intestinal epithelial cells (IECs) were analyzed by Western blot. (B) Western blot for RIOK1 in HCT116 and SW480 cells infected with shRIOK1, or control shRNA lentiviral vector, as well as in RKO and LOVO infected with the RIOK1-expressing or empty control vector (n = 4).And the intensity of the western blot bands was quantified using NIH ImageJ software. (C) Western blot for RIOK1 in the indicated HCT116 cells transfected with shRIOK1#1 or the shRNA-resistant expression construct, RIOK1Δ. RIOK1 expression was recovered in the HCT116-shRIOK1 cells transfected with RIOK1Δ (lower-panel).And the intensity of the western blot bands was quantified using NIH ImageJ software. RIOK1Δ almost restored the proliferative ability of the cells (upper-panel). (D) RIOK1 knockdown inhibited proliferation of HCT116 cells determined by CCK-8 and colony formation assays. (E) RIOK1 knockdown inhibited migration and invasion of HCT116 cells determined by transwell assays. (F) and (G) Ectopic expression of RIOK1 promoted proliferation of RKO cells determined by CCK-8 and colony formation assays. (H) Ectopic expression of RIOK1 promoted migration and invasion of RKO cells determined by transwell assays. (I) Representative immunofluorescence images demonstrated that RIOK1 level has an effect on the expression of EMT proteins in CRC cells. (J) Growth curve of subcutaneous injection of HCT116/Scramble and HCT116/shRIOK1 in NOD/SCID mice (n = 6 per group, left panel). NOD/SCID mice were injected subcutaneously into opposite flanks with 1.5 × 10⁶ control cells and RIOK1-knocked down HCT116 cells. The mice were sacrificed and the tumors were then removed, weighed and compared (right panel). And knockdown of RIOK1 in a xenograft tumor model was analyzed with western blotting, and the intensity of the western blot bands was quantified using NIH ImageJ software. (K) Growth curve of subcutaneous injection of RKO/Vector and RKO/RIOK1 in NOD/SCID mice (n = 6 per group, left panel). The tumors were then removed, weighed and compared (right panel). Over-expression of RIOK1 in a xenograft tumor model was analyzed with western blotting, and the intensity of the western blot bands was quantified using NIH ImageJ software. Effects of RIOK1 Knockdown (L) or overexpression (M) on lung metastasis of indicated cells in NOD/SCID mice (n = 10 per group): the number of metastatic nodules in the lung (left-panel); representative morphological observation of lung metastases (right-upper panel); and histopathological observation of lung sections (right-lower panel). The results are presented as are means ±SD (n = 3 for each panel). Statistical significance was concluded at *p<0.05, **p<0.01, ***p<0.001; # represents no statistical significance.

Figure 2—figure supplement 1. RIOK1 promotes the proliferation, invasion and metastasis of GC in vitro and in vivo.

(A) Western blot for RIOK1 in MKN45 cells infected with shRIOK1, or control shRNA lentiviral vector, as well as in SGC7091 infected with the RIOK1-expressing or empty control vector (n = 4).And the intensity of the western blot bands was quantified using NIH ImageJ software. (B) Western blot for RIOK1 in the indicated MKN45 cells transfected with shRIOK1#1 or the shRNA-resistant expression construct, RIOK1Δ. RIOK1 expression was recovered in the MKN45-shRIOK1 cells transfected with RIOK1Δ.And the intensity of the western blot bands was quantified using NIH ImageJ software. RIOK1Δ almost restored the proliferative ability of the cells. The effect of RIOK1 on proliferation of MKN45 and SGC7091 cells was determined by CCK-8 and (C) colony formation assays. (D and E) The effect of RIOK1 on migration and invasion of MKN45 cells was determined by transwell assays. (F) Growth curve of subcutaneous injection of indicated plasmids in NOD/SCID mice (n = 6 per group). The mice were sacrificed and the tumors were then removed, weighed and compared. And knockdown or overexpression of RIOK1 in a xenograft tumor model was analyzed with western blotting, and the intensity of the western blot bands was quantified using NIH ImageJ software. (G) Effects of RIOK1 knockdown or over-expression on tumor metastasis of indicated cells in NOD/SCID mice (n = 10 per group): the number of metastatic nodules in the lung. The results are presented as are means ±SD (n = 3 for each panel). Statistical significance was concluded at *p<0.05, **p<0.01, ***p<0.001; # represents no statistical significance.

Figure 2—figure supplement 2. PI3K/AKT pathway is required for RIOK1-mediated CRC and GC cell proliferation and migration.

(A) CRC and GC cells were transfected with RIOK1 plasmid, and treated with LY29004 (30 μM) or A443654 (1 μM). Then cell proliferation (A1), migration(A2) and invasion (A3) were determined. (B) The whole-cell lysates in (A) were analyzed by Western blot with indicated antibodies. (C) The RIOK1-knockdown-HCT116 and MKN45 cells were transfected with indicated plasmids. The cell proliferation (C1), migration(C2) and invasiveness (C3) was tested. (D1 and D2) The cells in (C) were harvested at 48 hr for western blot analysis with indicated antibodies. The data represent the mean ±SEM from three independent experiments. (E1) Immunohistochemistry staining, (E2) correlation analysis and (E3) WB analysis for RIOK1 and phospho-AKT-Ser473 in human CRC and GC tissues(n = 20).