Fig 2: Force induced autophagy in PDLSCs modulates M1 macrophage polarization through AKT signaling. (A) Western blot of the expressions of TNF-a, Arg-1 and NF-?B/P65 by the application of force-loaded supernatant (FS) with rapamycin. AKT signaling activator IGF1 or equal volume of DMSO was added into supernatant. The protein levels were quantified. Results are presented as mean ± SD. n = 3. *P < 0.05, **P < 0.01 versus the left column. ##P < 0.01 versus middle column. (B) Western blot of the expressions of TNF-a, Arg-1 and NF-?B/P65 by the application of force-loaded supernatant (FS) with 3-MA. AKT kinase inhibitor GSK690693 or equal volume of DMSO was added into conditioned medium. Results are presented as mean ± SD. n = 3. *P < 0.05, **P < 0.01 versus the left column. #P < 0.05, ##P < 0.01 versus middle column. (C) Relative mRNA expression of M1/M2-related genes of THP-1-derived macrophages cultured with different conditions, including FS, FS + Rapa + DMSO and FS + Rapa + IGF1. (D) Relative mRNA expression of M1/M2-related genes of THP-1-derived macrophage cultured with different conditions, including FS, FS + 3 - MA + DMSO and FS + 3 - MA + GSK690693 groups. Results are presented as mean ± SD. n = 3–4. **P < 0.01 versus left column; #P < 0.05, ###P < 0.001 versus middle column.

Fig 3: Western blots of the proteins involved in Rho-Rac signaling at E14 in wild type (WT) and R6/2 mice. Densitometry was used to quantify the ratio of phosphorylated to total protein expression of a, b ROCK2, c, d PAK, e, f LIMK1, g, h Cofilin, i, j SSH1L, k, l Profilin. Each value was initially normalized to expression of a-tubulin in the corresponding lane. Note that the same a-Tubulin blot might appear in more than one panel because of re-probing for multiple targets with non-overlapping molecular weights on a given blot. The data are presented as mean ± sem (n = 3). *P < 0.05; **P < 0.01, between indicated groups

Fig 4: AIM2 inhibits the microglial antiviral pathway–related inflammation by suppressing the DNA-PK–AKT3 signaling axis. (A) ELISA analysis of TNF-a, IL-6, CCL2, and IFN-ß in the supernatants of WT and AIM2-/- microglia transfected with poly(dA:dT) for 8 h. (B) Immunoblot analysis of p-IRF3(S385), p-IRF3(S396), IRF3, p-TBK1, TBK1, p-AKT, AKT, p-AKT1, AKT1, p-AKT2, AKT2, AIM2, and ß-actin (loading control) in WT and AIM2-/- microglia transfected with poly(dA:dT) for 4 and 8 h. (C) WT and AIM2-/- microglia were transfected with siRNAs to silence Akt1 and Akt2 for 48 h, and then were transfected with poly(dA:dT) for 4 and 8 h. Cell lysates were immunoprecipitated (IP) with anti-AKT3 antibody followed by immunoblotting with p-AKT and AKT3. (D) Immunofluorescent labeling of p-IRF3(S385) (green) and CD11b (red) in the spinal cord of EAE-induced WT mice at day 18 after immunization. The merge of p-IRF3(S385) with CD11b is indicated by arrowheads. Scale bar, 50 µm. (E) WT and AIM2-/- microglia were transfected with poly(dA:dT) for 8 h. Then cell lysates were immunoprecipitated with anti-DNA-PK antibody followed by immunoblotting with DNA-PKcs, TBK1, IRF3, AKT3, and AIM2. (F) WT and AIM2-/- microglia were treated with DNA-PK inhibitor NU7441 (1 nM) and then were transfected with poly(dA:dT) for 8 h. The production of indicated cytokines and chemokines was analyzed by ELISA. (G) WT and AIM2-/- microglia were treated with DNA-PK inhibitor NU7441 (1 nM) and then were transfected with poly(dA:dT) for 8 h. The expression of p-IRF3(S385), p-IRF3(S396), IRF3, p-AKT, AKT, and ß-actin (loading control) was analyzed by immunoblot. (H) WT and AIM2-/- microglia were transfected with siRNAs to silence Akt1 and Akt2 for 48 h, and then treated with DNA-PK inhibitor NU7441 (1 nM) followed by poly(dA:dT) transfection for 8 h. Cell lysates were immunoprecipitated with anti-AKT3 antibody followed by immunoblotting with p-AKT and AKT3. Data are pooled from three independent experiments for A and F. Data are representative of three (B and G) or two (C, E, and H) independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars show mean ± SEM. Unpaired t test. NT, no treatment.

Fig 5: AR is regulated by Akt1 in the castration-resistant prostate cancer cell line. (A) Western blot analysis of p-Akt1, total Akt1, AR, PSA and FKBP5 expression in C4-2 cells transfected with miR-149 mimic and miR-149 inhibitor. (B) Semi-quantification of western blotting results demonstrated the expression of p-Akt1/total Akt1, AR/ß-actin, PSA/ß-actin and FKBP5/ß-actin. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. Akt1 NC + miR-149 mimic NC group. AR, androgen receptor; miR, microRNA; p-, phosphorylated; NC, negative control; FKBP5, peptidyl-prolyl cis-trans isomerase FKBP5; KD, knockdown.