PMC

SW1116 cell lines were subcutaneously inoculated into nude mice. Representative photographs show dissected xenograft tumors after mice were sacrificed A. Tumor weight at the end of the experiment B. Data are presented as means ± SEM. *P<0.05; **P<0.01. Representative immunohistochemical staining with anti-Ki-67 and anti-MAFB in xenograft tumors (bars: 100μm) C.

RNA was extracted from SW1116 cells transfected with scramble shRNA (SHC002), MAFB shRNA678 or MAFB shRNA679 and cell cycle factors controlling the G1/S phase transition were analyzed via RT-qPCR A. Schematic of the human CDK6 promoter regions B. Relative CDK6 promoter activity was analyzed by luciferase assay C. The pRL-SV40 vector and pGL4.15 luciferase reporter vector containing the CDK6 promoter region were co-transfected with an MAFB expression vector or mock vector into 293T cells. Data are presented as means ± SEM. **P<0.01.

SW1116 and HCT116 cells were sorted by flow cytometry after infection with MAFB shRNA678, MAFB shRNA679 or scramble shRNA (SHC002) A. MAFB was detected by western blotting (WB), with tubulin as an internal control. Cell viability was indicated by absorbance at OD 450nm two h after CCK8 was added. Colony formation assay with SW1116 and HCT116 cells infected with MAFB shRNA678 or scramble shRNA (SHC002) B. Cells were cultured for 10 d and stained with crystal violet. Colonies were counted and analyzed. Cell cycle analysis of scramble shRNA (SHC002), MAFB shRNA678 and MAFB shRNA679 knockdown SW1116 cells by flow cytometry, showing the percentage of G0/G1 or S phase cells (upper panel) C. HCT116 and SW1116 results were the same (lower panel). Data are presented as means ± SEM. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Somatic MAFB alterations reproduced from TCGA database showing MAFB gene amplification in a majority of tumor types A. Altered MAFB levels occurred in 9% of CRC cases. MAFB was upregulated in CRC tissues B. MAFB levels in 61 paired CRC and matched adjacent non-tumor tissues were determined by RT-qPCR and normalized to GAPDH. Data are expressed as the log₂ fold change (ΔCt [CRC/Non.]), and significant MAFB upregulation was defined as a log₂ fold change >1 Ba. MAFB mRNA levels in: CRC and adjacent non-tumor tissues Bb. different stage CRC tissues Bc. and CRCs with and without metastases Bd. MAFB immunohistochemical staining in human CRC tissues and paired adjacent non-tumor tissues C. Strong positive MAFB expression in CRC Ca. weak positive MAFB expression in CRC Cb. negative MAFB expression in CRC Cc. and negative MAFB expression in non-tumor colorectal tissue Cd. bars: 100μm. Data are presented as means ± SD. N.S. (non-significant), P≥0.05; *P<0.05; ****P<0.0001.

Representative cell cycle distribution images in the indicated SW1116 cell types as analyzed by flow cytometry A. Western blotting (WB) was performed to detect MAFB in the indicated SW1116 cell lines B. Percentage of the indicated SW116 cells in G0/G1 or S phase C. Schematic of MAFB binding to the human CDK6 promoter regions (left panel), Relative CDK6 promoter activity as analyzed by luciferase assay (right panel), The pRL-SV40 vector and pGL4.15 luciferase reporter vector containing CDK6 promoter regions were co-transfected into 293T with wild-type MAFB and MAFB^K32R expression vector or mock vector D. ChIP-qPCR analysis of MAFB binding to the CDK6 locus, The MAFB-shRNA678 SW1116 cell line was transfected with Flag-MAFB or Flag-MAFB^K32R, and ChIP-qPCR was performed with the primer sets indicated in the upper diagram E. Data are presented as means ± SEM. **P<0.01; ****P<0.0001.

Flag-MAFB was co-transfected into 293T cells with HA-SUMO1 or mock vector, then immunoprecipitated by anti-Flag Gel and examined by western blotting (WB) for the presence of Flag-MAFB-SUMO1 conjugates A. Whole cell lyses (WCL) from A. were detected by WB with antibodies against Flag and HA B. 293T cells were co-transfected with the indicated vectors (top), and cell lysates were immunoprecipitated with Flag-Gel or HA-Gel C. The in-put and precipitates were examined by WB with antibodies against Flag and HA. The indicated four lysine (K) residues were replaced with an arginine (R) through point mutagenesis D. Flag-MAFB wild-type and K32R mutants were transfected with HA-SUMO1 vector into 293T cells. Cell lysates were immunoprecipitated by anti-Flag Gel and examined by WB with antibodies against Flag and SUMO1. Whole cell lysates from D. were detected by WB with antibodies against HA, Flag and H3 E. GST-MAFB and GST-MAFB^K32R proteins were purified by GSH-magnetic beads and stained with Coomassie brilliant blue after polyacrylamide gel electrophoresis F. SUMOylation assay was performed with the indicated elements (top), and the reactions were analyzed by WB using antibodies against MAFB, SUMO1 and GST tag G. WB was performed using antibodies against MAFB (rabbit polyclonal antibody) and SUMO1 to detect MAFB-SUMO1 conjugates immuno-precipitated by goat polyclonal antibody MAFB from SW1116 or HCT116 cell lysates H.