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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: Vascul Pharmacol. 2016 May 14;83:47–56. doi: 10.1016/j.vph.2016.05.002

Fig. 5.

Fig. 5

STAT3 positively regulates HK2 expression in PDGF stimulated VSMC and its inhibition is connected with decreased glycolysis and migration. (A) VSMC were transfected with 200 nM siRNA (scr or targeting Nrf2), after 24 h recovery starved for 18 h and then either treated with I3MO (5 μM)/PDGF as indicated for 8 h (upper panel) or with the proteasome inhibitor MG132 (10 μM) for 4 h (lower panel). Cells were lysed and total cell lysates subjected to western blot analysis for Nrf2 or actin and hexokinase or tubulin, respectively. Representative blots and compiled densitometric data from three independent experiments are depicted. (n = 3, mean + SD.; * p < 0.05; ANOVA, Bonferroni). (B) Quiescent VSMC were pretreated with 50 and 100 μM of the STAT3 inhibitor SI-602 for 30 min and then exposed to PDGF (10 ng/mL) for 6 h. Total cell lysates were subjected to immunoblot analysis for HK2 and actin. Representative blots and compiled densitometric data are depicted. (n = 3, mean + SD.; * p < 0.05; ANOVA, Bonferroni). (C) VSMC were transfected with 0, 60 or 200 nM siRNA targeting STAT3 with a constant total siRNA concentration of 200 nM in all samples (filled up with scrambled siRNA where needed), starved and then treated with PDGF for 8 h. Cells were lysed and immunoblotted for STAT3, HK2 and actin. Representative blots and compiled densitometric data of three independent experiments are depicted. (D) Glycolytic activity in quiescent and PDGF-stimulated (6 h) VSMC in the presence and absence of the STAT3 inhibitor SI 602 (75 μM) was determined by extracellular flux analysis as described. The bar graph depicts compiled data of three independent experiments (n = 3, mean + SD.; * p < 0.05; Student’s t-test). (E) Serum-deprived VSMCs that were either transfected with 200 nM scrambled siRNA or siRNA targeted versus STAT3 were subjected to a wound healing (scratch) assay with or without stimulation with 10 ng/ml PDGF-BB. Graphs indicate relative values of wound closure as assessed by CellProfiler Software (n = 3, mean + SD.; * p < 0.05; ANOVA, Bonferroni).