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. 2017 Dec 14;6:e29750. doi: 10.7554/eLife.29750

Figure 5. The regulation of labile Fe(II) by cAMP is likely mediated via RapGEF2.

(A) Pretreatment with PKA inhibitors (KT5720 (2 μM), H89 (20 μM)), Epac inhibitor ESI09 (10 μM) or CNGC blocker LCD (10 μM) for 20 min prior to cAMP addition showed no effect on labile Fe(II) induced by cAMP (100 μM) treatment in Schwann cells detected by Trx-Puro probes. (B) Knocking down the expression of RapGEF2 in HEK-293 cells largely blocked the acidification of vesicles and labile Fe(II) elevation after cAMP (100 μM) treatment compared to the scramble siRNA group. (C) IF quantification indicates that knocking down RapGEF2 inhibits but does not completely abolish vesicle acidification induced by cAMP. (D) IF quantification indicates that knocking down RapGEF2 inhibits but does not completely abolish labile Fe(II) induced by cAMP. Scale bar = 20 μm. p<0.0005 (n = 3 independent experiments with three biological replicates in each group).

Figure 5—source data 1. Fragments per kilobase per million (FPKM) of CNG and Rapgef genes in Schwann cells.
elife-29750-fig5-data1.docx (68.8KB, docx)
DOI: 10.7554/eLife.29750.022

Figure 5.

Figure 5—figure supplement 1. Abolishment of PKA activity by inhibitors H89 and KT5720.

Figure 5—figure supplement 1.

(A) Pretreatment with H-89 (20 μM) and KT5720 (2 μM) decreased the band of phosphorylated Peptag peptide induced by cAMP (100 μM) treatment. (B) Quantification of the assay showing that H89 (20 μM) and KT5720 (2 μM) were sufficient to block the activity of PKA (n = 2 independent experiments with one biological replicates each).
Figure 5—figure supplement 2. qRT-PCR shows that the mRNA level of RapGEF2 is lower in the siRNA group compared to the scramble siRNA group (p=0.042) (n = 3 independent experiments with three biological replicates each, error bars denote standard error).

Figure 5—figure supplement 2.

Figure 5—figure supplement 3. RAP1 and labile Fe(II) induction by cAMP.

Figure 5—figure supplement 3.

(A) Knocking down the expression of Rap1 largely blocked the effect of cAMP (100 μM) on vesicle acidification and labile Fe(II) elevation detected by Trx-Puro probes, while knocking down RAP2 and scramble siRNA had no obvious effect. Scale bar = 20 μm. (B) IF quantification indicates the inhibition of RAP1 siRNA on cAMP-induced vesicle acidification. (C) IF quantification indicates the inhibition of RAP1 siRNA on cAMP-induced labile Fe(II). (E) qRT-PCR shows the mRNA level of Rap isoforms are lower in the siRNA group compared to the scramble siRNA group. *p<0.005 (n = 1 independent experiment with three biological replicates each, error bars denote standard error).