Figure 4
Figure 4. Increased transformation potential associated with of Rpl22 loss or inactivation is accompanied by induction of Lin28B. Effect of Rpl22 knockdown on Lin28 expression in immortalized MEF. MEF lines stably expressing control or 2 different Rpl22 shRNA constructs were harvested for RNA and protein, after which Lin28A and Lin28B mRNA levels were evaluated by real-time PCR (A). **P < .005 for Lin28B expression in Rpl22 shRNA relative to controls. Protein levels were measured by blotting (B). Data are representative of 2 independent experiments. (C) Let-7 miRNA levels in immortalized MEFs where Rpl22 expression was suppressed by shRNA. Expression of Let-7 family miRNA was evaluated by real-time PCR in immortalized MEFs stably expressing control or Rpl22 shRNA constructs. Expression levels were normalized to sno202 RNA and to the expression level in cells transduced with control shRNA. Mean expression levels of triplicate measurements ± SD are represented graphically. Data are representative of 3 independent experiments. P < .05 for Let-7 miRNA levels in Rpl22 shRNA compared with control shRNA. (D) Effect of Rpl22 knockdown on expression of Let-7 targets. Expression of Ras and Myc was assessed by immunoblotting in control or Rpl22 knockdown MEFs. GAPDH served as a loading control. Expression of Lin28B in primary MEFs. Lin28B protein and mRNA levels were measured in primary MEFs of the indicated genotypes by immunoblotting (E) and real-time PCR (F), respectively. Mean ± SD of Lin28B mRNA expression levels are depicted graphically. Results are representative of at least 3 experiments performed. *P < .05. (G) Lin28B expression in primary thymocytes. The expression of Lin28B in thymocytes from mice with the indicated genotypes was evaluated by immunoblotting. GAPDH served as a loading control. Lin28B expression in MEFs after knockdown of Rpl11 and Rpl24. Immortalized MEFs were transduced with 2 different shRNA constructs targeting Rpl24 or Rpl11, after which protein levels were evaluated by immunoblotting (H). GAPDH served as loading control. (I) The level of Lin28B mRNA expressed by these cells was measured by real time PCR as in panel F. **P < .005.

Increased transformation potential associated with of Rpl22 loss or inactivation is accompanied by induction of Lin28B. Effect of Rpl22 knockdown on Lin28 expression in immortalized MEF. MEF lines stably expressing control or 2 different Rpl22 shRNA constructs were harvested for RNA and protein, after which Lin28A and Lin28B mRNA levels were evaluated by real-time PCR (A). **P < .005 for Lin28B expression in Rpl22 shRNA relative to controls. Protein levels were measured by blotting (B). Data are representative of 2 independent experiments. (C) Let-7 miRNA levels in immortalized MEFs where Rpl22 expression was suppressed by shRNA. Expression of Let-7 family miRNA was evaluated by real-time PCR in immortalized MEFs stably expressing control or Rpl22 shRNA constructs. Expression levels were normalized to sno202 RNA and to the expression level in cells transduced with control shRNA. Mean expression levels of triplicate measurements ± SD are represented graphically. Data are representative of 3 independent experiments. P < .05 for Let-7 miRNA levels in Rpl22 shRNA compared with control shRNA. (D) Effect of Rpl22 knockdown on expression of Let-7 targets. Expression of Ras and Myc was assessed by immunoblotting in control or Rpl22 knockdown MEFs. GAPDH served as a loading control. Expression of Lin28B in primary MEFs. Lin28B protein and mRNA levels were measured in primary MEFs of the indicated genotypes by immunoblotting (E) and real-time PCR (F), respectively. Mean ± SD of Lin28B mRNA expression levels are depicted graphically. Results are representative of at least 3 experiments performed. *P < .05. (G) Lin28B expression in primary thymocytes. The expression of Lin28B in thymocytes from mice with the indicated genotypes was evaluated by immunoblotting. GAPDH served as a loading control. Lin28B expression in MEFs after knockdown of Rpl11 and Rpl24. Immortalized MEFs were transduced with 2 different shRNA constructs targeting Rpl24 or Rpl11, after which protein levels were evaluated by immunoblotting (H). GAPDH served as loading control. (I) The level of Lin28B mRNA expressed by these cells was measured by real time PCR as in panel F. **P < .005.

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