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. Author manuscript; available in PMC: 2012 Apr 15.
Published in final edited form as: Biochem Pharmacol. 2011 Jan 26;81(8):1016–1027. doi: 10.1016/j.bcp.2011.01.006

Figure 2. Overexpression of inactive mutants or siRNA-Arf6 or siRNA-ARNO markedly inhibits glucose-induced insulin secretion in INS 832/13 cells.

Figure 2

Figure 2

Figure 2

Panel A: INS 832/13 cells were either mock-transfected or transfected with dominant mutants of Arf6 [T27N] or ARNO (E156K; see Methods). Relative degrees of expression of the mutants was verified by Western blotting. A representative blot from three independent studies yielding similar results is provided here.

Panel B: INS 832/13 cells were either mock-transfected or transfected with siRNA-Arf6 or siRNA-ARNO as described in the Methods section. Relative degrees of knockdown of these proteins were verified by Western blotting. A representative blot from three independent studies yielding similar results is shown here.

Panel C: INS 832/13 cells were transfected with dominant negative Arf6 [T27N] at a final concentration of 0.2 μg of DNA and cultured for 48 hr. Following this, cells were stimulated with either low [2.5 mM] or high [20 mM] glucose in KRB for 30 min at 37°C. Insulin released into the media was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly.

Panel D: INS 832/13 cells were transfected with dominant negative ARNO [E156K] at a final concentration of 0.2 μg of DNA and cultured for 48 hr following which cells were stimulated with either low [2.5 mM] or high [20 mM] glucose for 30 min at 37°C. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock transfected low glucose and **p < 0.05 vs. mock transfected high glucose, and data points with similar symbol do not differ significantly.

Panel E: INS 832/13 cells were either mock-transfected or transfected with Arf6-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells was stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly.

Panel F: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from five independent experiments. *represents p < 0.05 vs. mock transfected low glucose; **p < 0.05 vs. mock transfected high glucose. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly.

Panel G: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or KCl [60 mM; osmolarity adjusted] for 60 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. *represents p < 0.05 vs. mock-transfected low glucose; **p < 0.05 vs. mock transfected K+. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly.

Panels H and I: INS 832/13 cells were mock-tranfected or transfected either with Arf6-siRNA/ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with 1 mM glucose [Glu] or 1 mM glucose + 20 mM arginine [L-Arg] for 15 min. Insulin released into the medium was quantitated and expressed as percent of control. Data are mean ± SEM from replicates. * represents p < 0.05 vs. mock-transfected 1 mM glucose; **p < 0.05 vs. mock-transfected 1 mM Glu + 20 mM L-Arg. Insulin release values between mock or siRNA transfected under low glucose [1 mM] conditions did not differ significantly.