Figure 2

Effects ofETS1 siRNA-mediated knockdown on MMP9 protein expression in salivary gland derived cell lines. Semi-quantitative(densitometric) Western blotanalysis(A/B)and representative Western blots(C/D) of siRNA knockdown experimentsareshown. ETS1andMMP9 protein levels were determined 72hrspost-transfection with siRNA targetingETS1 (siETS1), relative to non-targetingsiRNA (siNT) (control)in HMC-3A(A/C)and A253(B/D)whole cell lysates. MMP9 protein levels were reduced in bothA253 and HMC-3A cells when transfected with 3 A cells when transfected with siETS1. Equal protein amounts were loaded into each lane and respective target normalized to cofilin protein expression. Western blot sections corresponding to either (ETS1 and Cofilin) or (MMP9 and Cofilin) were acquired from the same gel/blot, percell line, and processed identically from the same replicate sample, if possible. Loading controls and target proteins of the same gel/blotare imaged separately for optimized exposure requirements among the loading control vs. target protein (s). The effectsofETS1(siETS1) siRNA knockdown onMMP9 protein secretion into culture media were also determined (E).Total MMP9 (active and inactive) was measured by ELISA and presented as fold-decrease of the non-targeting siRNA (siNT) control. InSGCLs HMC-3A (n = 6) and A253 (n = 6), there were significant reductions in total MMP9 protein supernatant levels after siRNA knockdown of ETS1(E). Total MMP9 in the cell culture supernatant of iSGEC-pSS1 (n = 3) and iSGEC-nSS2 (n = 3) was significantly reduced by ETS1 knockdown (E). Mann–Whitney U-test was used to determine significant differences among the control (siNT) and siETS1. Results are presented as mean +/− standard deviation (SD). (***p < 0.001), (**p < 0.01), (*p < 0.05).