Figure 2.
Figure 2. NCL, through the regulation of a subset of miRNAs, affects specific changes in their target gene levels. (A) Luciferase assay using pGL3–miR-21–6X-BS (Sponge–miR-21) and pGL3–miR-103–4X-BS (Sponge–miR-103) reporter vectors after knockdown of the indicated genes in HeLa cells. pGL3–miR-30s–4X-BS (Sponge–miR-30a) was used as negative control. (B) Luciferase assay using pGL3-Sponge reporter vectors after knockdown of the indicated genes in HeLa cells and transfection with primary or mature miRNAs, as indicated. (A and B) All data are presented as mean ± SD of four independent experiments performed in triplicate. a.u., arbitrary units. (C) qRT-PCR analysis of the indicated miRNAs performed on MCF-7 cells after 72 h of transfection with NCL (si-NCL) or control (si-Scr) siRNA transfection. Values represent the mean ± SD from three independent sets of experiments. *, P < 0.01. (D) Relative quantification of DICER mRNA levels from HeLa cells transfected with scrambled siRNA (si-Ctrl) and si-NCL in the presence or absence of mature miR-103. Each data sample was normalized to the endogenous reference gene GAPDH by the 2−Δct method. (E) Luciferase assay on HeLa cells transfected with a reporter vector containing the 3′ UTR of DICER downstream the firefly luciferase gene along with the scrambled siRNA (si-Ctrl) and si-NCL in the presence or absence of mature miR-103. (D and E) Values represent the mean ± SD of three independent sets of experiments performed in triplicate. *, P < 0.01 compared with the si-Ctrl–transfected cells. (F and G) TIMP-3 (F) and FAS (G) protein levels in HeLa cells transfected with si-NCL or si-Ctrl with or without miR-221 or miR-21 rescue. β-ACTIN was used as loading control. Numbers below the blots indicate densitometric analysis showing TIMP-3/β-ACTIN and PTEN/ β-ACTIN ratios. (H) Expression of the indicated proteins by immunoblot analysis of HeLa cells transiently transfected with si-NCL or si-Scr in the presence or absence of miR-221/222. miR-26a was used as a PTEN-targeting, NCL-independent miRNA control. (F–H) Images are representative of at least three different experiments.

NCL, through the regulation of a subset of miRNAs, affects specific changes in their target gene levels. (A) Luciferase assay using pGL3–miR-21–6X-BS (Sponge–miR-21) and pGL3–miR-103–4X-BS (Sponge–miR-103) reporter vectors after knockdown of the indicated genes in HeLa cells. pGL3–miR-30s–4X-BS (Sponge–miR-30a) was used as negative control. (B) Luciferase assay using pGL3-Sponge reporter vectors after knockdown of the indicated genes in HeLa cells and transfection with primary or mature miRNAs, as indicated. (A and B) All data are presented as mean ± SD of four independent experiments performed in triplicate. a.u., arbitrary units. (C) qRT-PCR analysis of the indicated miRNAs performed on MCF-7 cells after 72 h of transfection with NCL (si-NCL) or control (si-Scr) siRNA transfection. Values represent the mean ± SD from three independent sets of experiments. *, P < 0.01. (D) Relative quantification of DICER mRNA levels from HeLa cells transfected with scrambled siRNA (si-Ctrl) and si-NCL in the presence or absence of mature miR-103. Each data sample was normalized to the endogenous reference gene GAPDH by the 2−Δct method. (E) Luciferase assay on HeLa cells transfected with a reporter vector containing the 3′ UTR of DICER downstream the firefly luciferase gene along with the scrambled siRNA (si-Ctrl) and si-NCL in the presence or absence of mature miR-103. (D and E) Values represent the mean ± SD of three independent sets of experiments performed in triplicate. *, P < 0.01 compared with the si-Ctrl–transfected cells. (F and G) TIMP-3 (F) and FAS (G) protein levels in HeLa cells transfected with si-NCL or si-Ctrl with or without miR-221 or miR-21 rescue. β-ACTIN was used as loading control. Numbers below the blots indicate densitometric analysis showing TIMP-3/β-ACTIN and PTEN/ β-ACTIN ratios. (H) Expression of the indicated proteins by immunoblot analysis of HeLa cells transiently transfected with si-NCL or si-Scr in the presence or absence of miR-221/222. miR-26a was used as a PTEN-targeting, NCL-independent miRNA control. (F–H) Images are representative of at least three different experiments.

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