Fig 2: Activation of mTORC2 engenders structural remodelling of nociceptive distal endings in epidermis.A-443654 (20 µL of 8 mM, intraplantar) was injected in ControltdTom (A) and Rictor-cKOtdTom (B) mice, and glabrous skin from the injected paw was collected 24 hours later. Representative images of the low (top) and high (bottom) magnification of nociceptive fibers in the epidermis, acquired using confocal microscopy with AiryScan. A-443654 increases main fiber length in ControltdTom but not Rictor-cKOtdTom mice (C, n = 5 mice per group). Similarly, A-443654 increases the number of branches per fiber in ControltdTom but not Rictor-cKOtdTom mice (D, n = 5 mice per group). ControltdTom (E) and Rictor-cKOtdTom (F) mice were injected with CFA (intraplantar), and glabrous skin from the injected paw was collected at day 1, day 3, day 7, and day 14 after injection. CFA increases main fiber length in ControltdTom but not Rictor-cKOtdTom mice at day 1 after CFA (G, n = 4–5 per group). Similarly, CFA increases the number of branches per fiber in ControltdTom but not Rictor-cKOtdTom mice (H, n = 4–5 per group). Statistics are based on 2-way ANOVA followed by Bonferroni’s post hoc comparison. All data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars: 20 µm.

Fig 3: Rictor knockdown promotes cell cycle arrest. (A) Cell cycle progression analysis by flow cytometry showed G0/G1 phase arrest in sh-Rictor cells. (B) Quantification of the cell cycle, Data are presented as the mean ± SD of independent experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 vs control.

Fig 4: Docetaxel-resistance reversal effect of Rapamycin and AZD8055 on cell proliferation. (A) IC50 was detected by cell counting kit-8 (CCK-8). Docetaxel-resistant PC-3 cells treated with increasing concentrations of Rapamycin or AZD8055 respectively. (B) Protein expression of Rictor and Raptor of PC-3/DTX cells treated with Rapamycin or AZD8055 at the concentration of IC50 for 48h respectively. (C) After the 48h treatment of Rapamycin or AZD8055 at the concentration of IC50, cells then treated with increasing concentrations of Docetaxel, and CCK-8 assay was used to test the change of IC50 to docetaxel between groups. Data are presented as the mean ± standard deviation of independent experiments performed in triplicate. **P<0.01, ***P<0.001 vs control.

Fig 5: mTORC1 inactivation increases SV replenishment rate.(A1) Cumulative EPSC charge in Raptor-Con (black) and Raptor-KO (red) neurons. Circles are the population means and the dotted lines are the s.e.m. The solid lines are linear fits to the final 20 responses and the y-intercept indicates the RRPtrain. (A2) Release probability calculated using RRPtrain is unchanged in Raptor-KO neurons. (A3) The SV replenishment rate is significantly increased by Raptor loss. (B1) Cumulative EPSC charge in Rictor-Con (black) and Rictor-KO (blue) neurons. (B2) Release probability calculated using RRPtrain is significantly decreased in Rictor-KO neurons. (B3) The SV replenishment rate is unaffected by Rictor loss. (C1) Example traces of normalized current responses to 500 mM sucrose application in Raptor-Con (black) and Raptor-KO (red) neurons. The black line shows the pre-sucrose baseline, and the gray shaded area shows the area used to calculate the replenishment rate. (C2) Plot showing the rate constants for sucrose-induced synaptic vesicle (SV) release in Raptor-Con (black) and Raptor-KO (red) neurons. (C3) Plot showing the replenishment rate constants after sucrose-induced SV release in Raptor-Con (black) and Raptor-KO (red) neurons. (D1) Example traces of normalized current responses to 500 mM sucrose application in Rictor-Con (black) and Rictor-KO (blue) neurons. The black line shows the pre-sucrose baseline, and the gray shaded area shows the area used to calculate the replenishment rate. (D2) Plot showing the rate constants for sucrose-induced SV release in Rictor-Con (black) and Rictor-KO (blue) neurons. (D3) Plot showing the replenishment rate constants after sucrose-induced SV release in Rictor-Con (black) and Rictor-KO (blue) neurons. In the dot plots, each dot represents the mean value from one neuron obtained from three independent cultures per genotype, and the bars show the estimated marginal means and s.e.m. ***=p < 0.001, and n.s. = p > 0.05, as tested with Generalized Estimating Equations.