Fig 2: The PI3Kγ signaling is enhanced in the human heart tissue with Chagas disease. a Transcriptome analysis of family I PIK3 genes expression (PIK3CA, PIK3CB, PIK3CD, and PIK3CG) in healthy control (Ctl; n = 7) and patients with idiopathic dilated cardiomyopathy (DCM; n = 14) or chronic chagasic cardiomyopathy (CCC; n = 10). b RT-PCR analysis of the mRNA expression of PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes in the heart tissue of Ctl (n = 5); DCM (n = 10); and CCC (n = 10) patients. GAPDH was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of Ctl (n = 5), DCM (n = 10), and CCC (n = 10) patients. GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of Ctl (n = 5), DCM (n = 10), and CCC (n = 10) patients. GAPDH was used as a loading control. AU refers to arbitrary units. ns = no statistical significance (one-way ANOVA with Tukey’s post hoc test in b–d). *P < 0.05 and **P < 0.01 relative to Ctl and #P < 0.05, ##P < 0.01, and ###P < 0.001 relative to DCM (one-way ANOVA with Tukey’s post hoc test in b–d). Data are from one experiment (mean ± s.e.m in b–d)

Fig 3: AKT1 is required for cGAS/STING function independent of Palbociclib-induced senescence. (A). Western blot of cGAS and IRF3 phosphorylation 48 h after transfection with 1 μg/mL dsDNA (G3-YSD). (B,C). Induction of IFNα/β by RT-qPCR 48 h after transfection with G3-YSD. (D). ELISA for 2′3′-cGAMP from lysates of transfected cells. (E,F). Induction of IFNα/β by RT-qPCR after 7 days of treatment with Palbociclib (PALBO) at 1 μΜ, PALBO with cGAS inhibitor (Ru521) at 2.5 μΜ, or PALBO with STING inhibitor (H151) at 5 μΜ (G,H). Beta-galactosidase staining of cells treated for 7 days with PALBO (1 μΜ) alone or in combination with cGAS inhibitor Ru521 (2.5 μΜ) or STING inhibitor H151 (5 μΜ). (I–K). SASP induction after treatment with PALBO and cGAS/STING inhibitors. Error bars represent SEM. Significance determined by Student’s t-test (Panel (B–D)) or two-way ANOVA with multiple comparisons (Panel (E–K)) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, n.s. = no significance). The uncropped blots are shown in Supplementary Materials.

Fig 4: mTOR signaling is deactivated during differentiation and only partially contributes to the control of proliferation.(a) Increasing densities of HaCaT were seeded to promote differentiation and harvested after 72h. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies. (b) NHK were serum-starved and differentiation was induced with 2mM CaCl2 for the indicated time points. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies was performed. Below a densiometrical quantification of involucrin and P-S6 levels of n = 4–5 similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (* p ≤0.05, ***p ≤0.001). (c) Keratinocytes stem cells (KSC), transient amplifying (TA) and postmitotic (PM) cells were separated according to their ability to adhere to type IV collagen. Protein lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies, showing that mTORC1 signaling is mainly present in undifferentiated cells. (d) Normal human skin was stained with P-mTOR S2448 and Ki-67 antibody. Nuclei were stained with DAPI. Single color and overlay images are presented, which show that mTOR is activated in proliferation cells of the basal layer. Bars represent 100 μm. (e) HaCaT cells were reverse-transfected with siRNA targeting Akt, Raptor or control siRNA and seeded in 96 well plates. After 48h proliferation was quantified using the XTT-based assay. Graph presents mean ± SEM (n = 4–8). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (f) To control for the efficiency of knockdown, HaCaT cells were transfected as in (e) and seeded in 6 well plates. Protein lysates were harvested after 48h and a Western blot was performed with the indicated antibodies. To show the consequences of Akt and Raptor knockdown, phosphorylation of the downstream targets GSK-3 and S6 was also captured. (g) HaCaT cells were seeded in 96 well-plates in triplicates and after 24h 50 μM LY294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) were added. After another 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (h) To show efficiency of the used inhibitors, HaCatT cells were treated as in (g). Protein lysates were prepared and a Western blot was performed with the indicated antibodies.

Fig 5: miR-16 treatment and Fluoxetine treatment was able to significantly increase PI3K/Akt/mTOR activity. (A) Images of mTOR Immunofluorescence staining show a decrease expression level in the CUMS groups compared with the Control groups. (B) Mean change in mTOR expression detected by immunofluorescence. (C) Compared with the Control groups, the protein expressions of mTOR, PI3K, and AKT levels decreased in the CUMS groups. Histograms from mTOR protein level (D), AKT protein level (E) and PI3K protein level (F) show significant restoration toward control levels in both miR-16 and Fluoxetine groups. Values are the means ± SEMs. *P < 0.05; **P < 0.001; ***P < 0.0001.