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. 2022 Nov 29;42(12):e00271-22. doi: 10.1128/mcb.00271-22

FIG 6.

FIG 6

Ago1 and Ago2 are involved in VINK transcriptional repression by VINK siRNA. (A) Senescent WI38 cells were recovered and subjected to fractionation experiments. Whole-cell extracts (WCE) and cytoplasmic (cyto), nuclear soluble (NS), and chromatin (CHR) fractions from the same amount of cells were analyzed by Western blotting using the indicated antibodies. (B) Senescent WI38 cells were transfected with 30 nM of the indicated siRNAs. At 72 h later, cells were harvested and total cell extracts were subjected to Western blot analysis with antibodies directed against Ago1, Ago2, or GAPDH as a loading control. The asterisks indicate nonspecific bands. Identical results were obtained in three independent experiments. (C) Senescent WI38 cells were transfected with 30 nM of the indicated siRNAs. The amount of siRNAs was kept constant at 60 nM, using Ctle-1 siRNA. At 72 h later, total RNA was prepared and analyzed by RT-qPCR for the expression of GAPDH, Ago1, or Ago2 mRNA or VINK. The amount of Ago1, Ago2, or VINK was standardized to GAPDH mRNA and calculated relative to a value of 1 for cells transfected with the Ctle-1 siRNA. The means and standard deviations from 3 independent experiments are shown. *, P < 0.05 comparing the effect of siVINK-1 in cells depleted for Ago1 or Ago2 to its effect alone (paired Student's t test). (D) Senescent WI38 cells were transfected with 25 nM of the indicated siRNAs. The amount of siRNAs was kept constant at 75 nM using Ctle-1 siRNA. At 72 h later, total RNA was prepared and analyzed by RT-qPCR for the expression of GAPDH, Ago1, or Ago2 mRNA or VINK. The amount of Ago1 or Ago2 mRNA or VINK was standardized to GAPDH mRNA and calculated relative to a value of 1 for cells transfected with the Ctle-1 siRNA. The means and standard deviations from 6 independent experiments are shown. *, P < 0.05 when comparing the effect of siVINK-1 in cells depleted for Ago1 and Ago2 to its effect alone (paired Student's t test). (E) Same experiment as in panel D, except that nascent RNAs were recovered. The amount of VINK was standardized to GAPDH and calculated relative to a value of 1 for cells transfected with the Ctle-1 siRNA. The means and standard deviations from three independent experiments are shown. *, P < 0.05 when comparing the effect of siVINK-1 in cells depleted for Ago1 and Ago2 to its effect alone (paired Student's t test).