Figure 3

TNFAIP8 regulates cell proliferation and migration in skin cancer cells. (A) Western blotting analysis of TNFAIP8-Myc tagged protein expression in HaCaT and skin cancer cells. Immunoreactive bands were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (B) Effect of TNFAIP8-Myc protein overexpression on HaCaT, A431, A375 and, A2058 on cell survival was measured by MTT assay. Results are representative of three independent experiments. *P < 0.05 relative to EV transfected cells. (C) The effect of overexpression of TNFAIP8-Myc on cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. *P < 0.05 relative to EV transfected cells. (D) HaCaT, A431, A375 and, A2058 cells were transfected with control siRNA or TNFAIP8 siRNA (100 nM) for 30 h, and cell lysates were western blotted with anti-TNFAIP8 and β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. TNFAIP8 and β-actin protein levels were quantified using ImageJ software (https://imagej.nih.gov/ij/) and presented below. (E) The effect of TNFAIP8 knockdown by siRNA on cell survival was analyzed by MTT assay. The experiments were independently performed three times. *P < 0.05 relative to control siRNA transfected cells. (F) Control siRNA and TNFAIP8 siRNA-transfected HaCaT, A431, A375 and A2058 cells (3000 cells/well) were re-plated in 6-well plates in triplicate for 7–10 days and skin cancer cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. *P < 0.05 relative to control siRNA transfected cells. (G) Wound healing assay: HaCaT, A431, A375 and A2058 cells were transfected with control siRNA or TNFAIP8 siRNA for 48 h. The effect of the TNFAIP8 knockdown on cell migration was analyzed using the wound healing assay. Representative images of the wound healing assay (top) and the calculated scratch area (bottom) are shown. *P < 0.05 relative to control siRNA transfected cells.