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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: RHBDD1 promotes colorectal cancer metastasis through the Wnt signaling pathway and its downstream target ZEB1

Fig. 2

RHBDD1 could affect the metastatic ability of colorectal cancer cells in vivo and in vitro. a, b. Images and statistics of the migration assay and invasion assay when RHBDD1 is knocked down in HCT-116, HCT-8 and DLD1 cells. For migration assay, 1 × 105 cells were seeded in the upper chamber of the transwell insert, as described in Methods section. For invasion assay, the upper chamber of the transwell insert was pre-coated with Matrigel to form the Matrigel layer in the chamber. A total of 2 × 105 cells were seeded in the upper chamber of the transwell insert as described in Methods section.× 100 magnification, cells were counted and calculated. Ctrl, control; Si-1#, Si-RHBDD1–1#; Si-2#, Si-RHBDD1–2#. c, d. Images and statistics of the migration and invasion assays when RHBDD1 is over-expressed in CACO-2 cells. RHBDD1 was cloned into a pcDNA6.0 plasmid with a C-terminal myc-tag. 2 μg plasmids were transfected for each well of 6-well cell culture plate. OE, over-expression. e, f. Images and statistics of the migration and invasion assays in HCT-116 cells expressing wild-type and mutant RHBDD1. WT, wild-type; MT, mutant. g, h. Images and statistics of the migration and invasion assays in HCT-116 cells when wild-type RHBDD1 expression is rescued. MT + Ctrl, mutant cell line infected with lentivirus-control; MT + OE, mutant cell line infected with lentivirus-RHBDD1. a-h. The experiments were performed in triplicate. i. In vivo imaging of metastasis in NOD/SCID mice injected with HCT-116 cells with stable knockdown of RHBDD1 or control Si-RNA. NOD/SCID mice were inoculated via tail vein injection (5 × 105 cells per mouse). Twenty-eight days later, the luciferase activity and the signal distribution in each mouse was detected. j. Statistical analysis of luminescence in i. The data in each column were calculated from 4 mice. All data are mean ± SEM, *, P < 0.05; **, P < 0.01; ***, P < 0.001, NS, not significant

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