Figure 6. siRNA to LCN2 specifically knocks down LCN2 expression in SEB-1 sebocytes in the presence of 13-cis RA.
SEB-1 sebocytes (2 × 106 cells per 100 μl reaction mixture) were nucleofected with 1 μg siCONTROL, GAPDH (as a specificity control), or LCN2 siRNA. 13-cis RA (0.1 μM) was added 24 hours after nucleofection, and cells were incubated for 48 and 72 hours. Extent of siRNA knockdown of gene expression was verified by QPCR and western blotting for LCN2 and GAPDH after 13-cis RA treatment. GAPDH and LCN2 gene expression were successfully inhibited in their respective samples. (A) QPCR analysis of LCN2 and GAPDH mRNA levels at 48 hours of 0.1 μM 13-cis RA treatment. The expression of LCN2 mRNA was successfully decreased 15-fold by the LCN2 siRNA compared with siCONTROL, whereas expression of GAPDH was minimally affected by siRNA to LCN2. GAPDH mRNA expression was decreased 4-fold by the specific GAPDH siRNA when compared with siCONTROL, whereas siRNA to GAPDH had minimal effects on expression of mRNA for LCN2. Data represent mean ± SEM; n = 6. *P < 0.05 as determined by REST-XL program. (B) Western analysis of GAPDH and NGAL protein levels. NGAL protein expression is decreased to undetectable levels by western blotting at 48 and 72 hours of 0.1 μM 13-cis RA treatment with LCN2 siRNA. Representative blots of 4 independent experiments are shown.