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. 2016 Jun 30;7(31):48887–48904. doi: 10.18632/oncotarget.10346

Figure 5. The availability of amino acids affects the response of rDNA transcription to growth factors.

Figure 5

A.-B. Exponentially growing HeLa cells (EXP) were starved of amino acids and serum (-AAS) for 2 hours and then stimulated with either amino acids (AA), 100 nM insulin (INS) or 10% dialyzed serum (DS), either alone or in combination as indicated, for 3 hours. Alternatively, HeLa cells were serum-starved (SS) for 24 hours then stimulated with 100 nM insulin or 10% dialyzed serum for 3 hours. A. Cells were pulse labeled and 47/45S rRNA synthesis analyzed. Representative images and resultant graph (mean +/− SEM) of n = 3 experiments. Below the graph are representative immunoblotting images, n = 2 experiments. B. qChIP analyses to assess Pol I loading on various regions of the rDNA. n = 3-5 experiments. C. HEK293 cells were treated as in A. Western blot analysis of immunoprecipitated ectopically expressed strep-HA-tagged RRN3 for endogenous POLR1B, and PAF53 in HEK293 cells. Representative images from n = 3 experiments.D. HeLa cells were amino acid and serum-starved for 2 hours, pre-treated with 10 μM PF4708671 for 30 minutes, and then stimulated with all amino acids for 3 hours. Cells were pulse labeled and 47/45S rRNA synthesis analyzed. Representative images and resultant graph (mean +/− SD) of n = 2 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.