Fig. 3: MAFF regulates tumor cell invasion.

a MAFF was knocked down using three different clones of shRNA against MAFF. Western blot data showed that while two of clones (C12 and D2) decreased MAFF expression significantly, one clone had little effect on MAFF expression (D1), which we decided to use as a negative control. b MAFF inhibition using shRNA did not affect tumor cell growth in MDA-MB-231 cells both under normoxia and hypoxia. n = 3 biological replicates. Unpaired t-test. c MAFF knockdown in MDA-MB-231 cells decreased tumor cell invasion through collagen (upper panels, n = 3 biological replicates) or Matrigel-coated transwell membranes (lower panels, n = 4 biological replicates). Quantification is included in graphs. One-Way ANOVA with multiple comparisons. d MAFF knockdown was rescued by overexpressing MAFF cDNA with mutations in the shRNA targeting region. Decreased tumor cell invasion by MAFF inhibition was reactivated when MAFF was re-expressed (mut: mutant). Tumor cell invasion was determined using collagen assay (upper panels) or Matrigel-coated transwell membrane assay (lower panels). n = 3 biological replicates. One-Way ANOVA with multiple comparisons. e MAFF overexpression in MCF7 cells did not show any significant difference in cell growth rates (ns: not significant). n = 3 biological replicates. Unpaired t-test. f Increased invasive phenotypes were observed in MAFF overexpressing MCF7 using collagen assay (upper panels, n = 3 biological replicates) and transwell invasion assay (lower panels, n = 4 biological replicates). Unpaired t-test.(Graphs represent the mean per group and error bars represent the SEM. ns: not significant).