Fig 2: mTORC1 is required for Akt induction of NF-?B in HNSCCA. Overexpression of Akt increases activity of mTORC1 and NF-?B. Different doses of active HA tagged Akt2 were transfected in Cal27 cells and phosphorylation p65 and S6K and total p65 and S6K, as well as HA-Akt2 expression were tested by western blot. B. Inhibition of Akt, mTORC1 and NF-?B by Akt inhibitor, perifosine. Cells were treated with different doses of perifosine for 24 hours and the levels of p65, Akt, S6K and of endogenous phosphorylation of S6K, p65, and Akt were determined by probing with the indicated antibodies. C. Depletion of Raptor blocks Akt induction of NF-?B. Cells were transfected with non-target siRNA or siRNA against Raptor as indicated for 48 hours and active Akt2 were transfected for another 24 hours. Cells were lysed and the levels of p65, Akt, S6K and of endogenous phosphorylation of S6K, p65, Akt2, and Raptor were determined. The results are representative of three experimental repetitions.

Fig 3: Akt cIEF assay development and peak identification. A, Western blot analysis of Akt isoforms (Akt1–3) in cell lysates obtained from N1E-115 neuroblastoma cells, primary cortical neurons (21DIV), and embryonic day 16.5 (E16.5) mouse brain. B, cIEF analysis of the cell lysates using a pan-Akt antibody shows a regular Akt profile with 9 or 10 conspicuous peaks that are separated according to protein charge distribution. C, cIEF analysis of cell lysates using isoform-specific Akt antibodies. In cIEF profiles, Akt1 peaks are colored in green, Akt2 peaks are in blue, and Akt3 peaks are in red. Peaks with mixed Akt isoforms are colored in yellow.

Fig 4: The RANTES-CCRs-Akt2 axis is activated in DDw/oR–induced early-stage TMJOA.Male Sprague-Dawley rats underwent right-sided unilateral DDw/oR surgery. Rats were euthanized at baseline (0 weeks), early (1 week), and late (8 weeks) time points after surgery. (A) Representative images of RANTES and CD68 immunofluorescence costaining in TMJ sagittal sections. RANTES+ cells appear in green and CD68+ cells appear in red. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate RANTES+ cells in articular cartilage. Scale bar: 50 µm. (B) Representative images of p-Akt2 and CD68 immunofluorescence costaining in TMJ sagittal sections. p-Akt2+ cells appear in green and CD68+ cells appear in red. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate p-Akt2 and CD68 double-positive cells in subchondral bone, and the asterisks indicate CD68 single-positive cells. Scale bar: 50 µm. (C) Quantitative analysis of the number of RANTES+ cells in articular cartilage of condyles. (D) Quantitative analysis of the number of CD68+ cells in subchondral bone of condyles. (E) Quantitative analysis of the ratio of p-Akt2+ and CD68 double-positive cells to CD68-positive cells in subchondral bone of condyles. Data are presented as mean ± 95% CI, and 1 representative image of 4 independent samples per group is shown. Statistical analyses were determined by 1-way ANOVA with Bonferroni’s multiple comparison test. **P < 0.01. Abbreviation: w, week.

Fig 5: Effects of a single heat stress and/or 14 days of denervation on the protein expression levels in sham-operated and denervated soleus muscles. The phosphorylated (p) and total (t) levels of Akt, Akt1, Akt2, and S6; the expression levels of HSP70 and GAPDH were evaluated by immune blotting. Representative results of immunoblotting are shown in A. The expression level of each protein was calculated after normalization using the expression levels of GAPDH (B, C, E, F, H, I, K, L, N). The phosphorylation rate of the protein was calculated by dividing the expression level of phosphorylated protein by the expression level of total protein (D, G, J, M). The relative values pertaining to the sham-operated Cont muscles are also shown. Means ± SD (n = 5 per group). H and D: main effect of heat stress and denervation, respectively; H×D: interaction of effects of heat stress and denervation; *: the result of Tukey's multiple comparisons test was P < 0.05. See Figure 2 and 4 for other abbreviations.