DNA damage induction in IHH cells by insulin treatment with or without PTEN inhibition.

a. Representative pictures for comets of the control (left) and of insulin-treated (right) IHH cells. b. Quantification of comet assay results. Data are presented as mean fold change compared to control ± SEM of 4 independent experiments. * p≤0.05 vs. HCl, Δ p≤0.05 vs. VO-OHpic, ○ p≤0.05 vs. 10 nM insulin, ● p≤0.05 vs. 100 nM insulin and □ p≤0.05 vs. VO-OHpic + 10 nM insulin.</p

Apoptotic and mitotic cells after treatment with insulin and the Pten-inhibitor VO-OHpic.

<p>Apoptotic and mitotic cells after treatment with insulin and the Pten-inhibitor VO-OHpic.</p

Relative expression of HSP70, HO-1, pAKT and pan-AKT in mouse liver.

<p><b>a.</b> Representative pictures of the bands from HSP70. <b>b.</b> Representative pictures of the bands from HO-1. <b>c.</b> Quantification: The band intensity of HSP70 (WT (ND, n = 6; HFD, n = 8), Pten +/- (ND, n = 9; HFD, n = 8) was quantified by image j. Data are presented as mean fold change compared to WT/ND ± SEM. * p≤0.05 and ** p≤0.01. <b>d.</b> Quantification: The band intensity of HO-1 (WT (ND, n = 5; HFD, n = 6), Pten +/- (ND, n = 6; HFD, n = 8) was quantified by image j. Data are presented as mean fold change compared to WT/ND ± SEM. * p≤0.05 and ** p≤0.01. <b>e.</b> Representative pictures of the bands from pAKT S473, pAKT T308 and pan-AKT.</p

MN induction in IHH cells by insulin treatment with or without PTEN inhibition.

a. Representative picture of micronuclei in a binucleated cell. b. Quantification of micronucleus test. Data are presented as mean fold change of MN-containing cells in 1000 binucleated cells (BN) compared to control ± SEM of 4 independent experiments. * p≤0.05 vs. solvent control, Δ p≤0.05 vs. VO-OHpic, ○ p≤0.05 vs. 10 nM insulin, ● p≤0.05 vs. 100 nM insulin. ▫ p≤0.05 vs. VO-OHpic + 10 nM insulin and ▪ p≤0.05 vs. VO-OHpic + 100 nM insulin.</p

Experimental timeline.

12-week-old C57BL/6 mice (WT and Pten +/-) were fed either with ND or with HFD for 20 weeks as described in “Materials and methods”.</p

Intracellular oxidative stress induction in IHH cells by insulin treatment with or without the PTEN inhibition.

<p><b>a.</b> Representative pictures for DHE staining. <b>b.</b> Quantification of DHE fluorescence by measuring the mean grey value of DHE signal from 250 cells using image j software. Data are presented as mean fold change compared to control ± SEM of 5 independent experiments. * p≤0.05 vs. solvent control, Δ p≤0.05 vs. VO-OHpic, ● p≤0.05 vs. 100 nM insulin and □ p≤0.05 vs. VO-OHpic + 10 nM insulin.</p

Cytokinesis block proliferation index (CBPI) after treatment with insulin and PTEN inhibitor VO-OHpic.

<p>Cytokinesis block proliferation index (CBPI) after treatment with insulin and PTEN inhibitor VO-OHpic.</p

Detection of ROS in mouse liver by DHE staining.

<p><b>a.</b> Representative pictures from DHE stained liver sections of Pten +/- and WT mice after feeding with ND or HFD. <b>b.</b> Quantification of ROS formation was achieved by measuring mean grey values of DHE signal from 200 cells using image j software. Mice (WT (ND, n = 6; HFD, n = 9), Pten +/- (ND, n = 9; HFD, n = 9)) had been fed with ND or HFD for 20 weeks. Data are presented as mean fold change compared to the WT/ND ± SEM. * p≤0.05 and ** p≤0.01.</p

Detection of γ-H2AX on paraffin embedded mouse liver.

<p><b>a.</b> A representative picture is shown as insert. The black arrows indicate γ-H2AX positive cells. <b>b.</b> Quantification of liver sections: 12 to 15 photos per animal were quantified by image j (WT (ND, n = 3; HFD, n = 8) and Pten +/- (ND, n = 8; HFD, n = 8). Data are presented as mean fold change of γ-H2AX positive cells compared to WT/ND ± SEM. * p≤0.05 and ** p≤0.01.</p

Viability of cells after treatment with insulin and the PTEN inhibitor VO-OHpic.

<p>Viability of cells after treatment with insulin and the PTEN inhibitor VO-OHpic.</p