Fig. 3

Fig. 3. SiRNAs mediated gene knockdown targeting terfa in ZF4. A.B. Knockdown efficiency of terfa of each scheme by RT-qPCR. A, 48 h after transfection; B, 72 h after transfection. C. Western blot of zfTRF2. Untreated and siRNAs transfected cells were prepared for WB after being transfected with X–HP for 72 h. D. SiRNAs mediated gene knockdown in ZF4 and ZFL. Knockdown efficiency of terfa was measured by RT-qPCR 48 h after transfection with 15 μL X-tremeGENE HP and 400 nM siRNAs in 6-well plate. E. SA-β-Gal assay of ZF4 transfected with siRNAs following trial-3 of X–HP at 72 h. Scale bar: 20 μm. F. Microscopy of untreated and transfected ZF4 cells. Results showed little cytotoxicity of Lipo3000 and X–HP and medium cytotoxicity of jetPrime, Effect a min and X-siRNA. scale bar: 50 μm. G. Off-target effect detection by RT-qPCR of shelterin genes terfa, terf1, pot1, rap1, tpp1, tin2 in ZF4 cells transfected with siterfa by X–HP 48 h later.

Lipo3000: Lipofectamin^TM 3000, X-siRNA: X-tremeGENE^TM siRNA, Effectene: Effectene® Transfection Reagent, X-HP: X-tremeGENE^TM HP.ns: none sense, ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001.