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. 2018 Jul 31;7:e36362. doi: 10.7554/eLife.36362

Figure 4. G2-PQS in PSPs form quadruplexes in Hela cells and self-regulate.

(a) Effects of pyridostatin (PDS) on spermine and spermidine (n=3, *p≤0.05, **p≤0.01). (b) Effects of PDS on endogenous SMS protein at 6 and 24 h (n=3, *p≤0.05, **p≤0.01) (full length blots are in Figure 4—figure supplement 4); effects on SMS5Q1 (n=3, *p≤0.05) and SMS3Q1 (n=3-5, **p≤0.01, ***p≤0.001) wild-type and mutant reporter gene expression (UT: untreated); (c) Effects of PDS on endogenous AZIN1 protein at 24 h (n=3, *p≤0.05) (full length blots are in Figure 4—figure supplement 4); effects on AZIN15Q1 wild-type and mutant reporter gene expression (n=6, *p≤0.05). (d) Effect of PA supplementation on reporter activity from PQS’s in HeLa cells (n=3, *p≤0.05, **p≤0.01). (e) Effect of DMFO and APCHA on levels of spermine and spermidine in HeLa cells. Two independent replicates were performed. (f) Effect of PQS’s from PSPs on reporter activity in HeLa cells under PA depletion (0.5 mM DFMO, 100 µM APCHA, 6 days) followed by PA rescue (0.5 mM DFMO, 100 µM APCHA, 6 days, 100 µM PA, addition day 5). (n=3-5, *p≤0.05). Error bars represent standard error (SE) from at least two independent replicates. See Figure 4—figure supplements 5, 6 and 7.

Figure 4.

Figure 4—figure supplement 1. Cell viabilities after PDS treatments of HeLa cells.

Figure 4—figure supplement 1.

Effects of the quadruplex stabilizing ligand Pyridostatin (PDS) on HeLa cell viability. The error represents the standard error (SE). (n = 3,**p≤0.01).
Figure 4—figure supplement 2. Effects of PDS treatment upon spermine and spermidine levels in HeLa cells.

Figure 4—figure supplement 2.

Example HPLC traces used for polyamine monitoring by HPLC at 229 and 198 nm, upon depletion of cellular polyamine levels following treatment with PDS for 24 hr (4, 16, 64 μM).
Figure 4—figure supplement 3. Effects of PDS treatment on PQS reporter genes in HeLa cells: un-normalized data.

Figure 4—figure supplement 3.

Effects of the quadruplex stabilizing ligand PDS on PQS-reporters in HeLa cells. Un-normalized Renilla data for the three reporters of Figure 4b and c after PDS treatment of cells.
Figure 4—figure supplement 4. Effects of PDS treatment on endogenous polyamine synthesis proteins in HeLa cells.

Figure 4—figure supplement 4.

Full-length blots.
Figure 4—figure supplement 5. Polyamine addition and depletion reporter assays.

Figure 4—figure supplement 5.

The error bars represent the standard error (SE) of at least two biological replicates. Normalized data.
Figure 4—figure supplement 6. Polyamine addition and depletion reporter assays.

Figure 4—figure supplement 6.

The error bars represent the standard error (SE) of at least two biological replicates. Un-normalized data.
Figure 4—figure supplement 7. Polyamine depletion and spermine synthase inhibition.

Figure 4—figure supplement 7.

Example HPLC traces used for polyamine monitoring by HPLC at 198, 229 and 254 nm, upon depletion of cellular polyamine levels following treatment with DFMO and APCHA. Confirmation of APCHA inhibition of spermine synthase under the conditions tested (putrescine or spermine addition).