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. Author manuscript; available in PMC: 2017 Dec 1.
Published in final edited form as: Cell Signal. 2016 Oct 2;28(12):1923–1932. doi: 10.1016/j.cellsig.2016.09.008

Fig. 4. The PKA/STAT3 signaling pathway mediates ADORA2b function in ePS cells.

Fig. 4

ePS cells from RM136 and RM159, grown in F-FM, were assessed for A. OCT3/4 transcripts by qRT-PCR in ePS cells treated or not with adenosine (Ado) in presence or absence of the PKA inhibitor H89 or the STAT3 inhibitor Stattic. (n=3 each). B. SON and STAT3 protein expression and phosphorylation status of STAT3 (p-STAT3) by WB in ePS cells with or without knock down of ADORA2b (shDORA2b), or treated with or without APCP for 48 h or treated with Ado for 48 h, in presence or absence of H89 (1 or 5μM) or Stattic (0.2 and 1μM) (n=1 and n=2, respectively). C. SON and STAT3 protein expression by WB after transduction of lentivirus carrying inducible shRNAs against STAT3 (#16 or #17) or a matched scrambled shRNA and induction by Doxycyclin for 48h (n=2 each). D. OCT3/4 transcripts by qRT-PCR in untreated ePS cells and ePS cells treated with Ado in presence or absence of H89 or Stattic, alone or various inhibitor combinations. Red dashed line: OCT3/4 expression in presence of H89, Stattic alone or their combination (n=3 each). E. OCT3/4 transcripts by qRT-PCR in MRS-treated or untreated ePS cells in presence or absence of the PKA activator 8-Br-cAMP (n=3 each). F. OCT3/4 protein expression and STAT3 phosphorylation status (phospho-STAT3) by WB in ePS cells treated or not with different concentrations of MRS-treated in presence or absence of different concentrations of 8-Br-cAMP (n=1 and n=2, respectively). G. IL6 and LIF transcripts by qRT-PCR in untreated or adenosine-treated ePS cells in presence or absence of H89 (n=3 each). H. IL6 transcripts by qRT-PCR in ePS cells treated or not with 10μm of 8-PT, MRS, DMPX and VUF (top panel). IL6 protein concentration by ELISA in CM collected from ePS cells with or without knock-down of ADORA2b (bottom panel) (n=2 each) I. IL6 transcripts by qRT-PCR in ePS cells treated or not with APCP in presence or absence of Ado or in ePS cells transduced with a CD73 shRNA or a scrambled shRNA (n=3 each). In Panels B, C and F loading control: ACTIN and data from RM136 are shown.