Fig 1: Association between Rictor and Cav 1 analyzed using western blot and apoptosis detection. (A, B) Two different cell lines were transfected with Rictor-siRNA respectively to verify Rictor-siRNA knock down efficiency. (C) Protein levels of p-Akt and Cav 1 induced by Rictor knockdown in SGC-7901 cells were assessed. (D) Protein levels of p-Akt and Cav 1 induced by Rictor knockdown in AGS cells. (E) After transfection of Rictor plasmid in SGC-7901 cells for 24 h and addition of 20 μM ly294002 for 6 h, the changes of p-Akt and Cav 1 protein levels were detected by western blot. (F) After transfection of Rictor plasmid into AGS cells for 24 h, and addition of 20 μM ly294002 for 6 h, the changes of p-Akt and Cav 1 protein levels were detected by western blot. β-actin served as loading control. (G) Apoptosis of SGC-7901 cells after 24 h transfection with Rictor plasmid and 24 h treatment with 20 μM ly294002 (n=3). (H) Apoptosis of AGS cells after 24 h transfection with Rictor plasmid and 24 h treatment with 20 μM ly294002 (n=3). Values represent the Means ± SD. **P < 0.01, ***P < 0.001 and ****P < 0.0001 were calculated using Student’s t-test.
Fig 2: Rictor is overexpressed in invasive melanoma and correlated with VM. (A), IHC staining of (a) Rictor, (c) AKT, (e) MMP‐2 expression. b, d, f were negative controls without primary antibodies for Rictor, AKT, MMP‐2, respectively. (B) Phenomena of VM (red arrow, CD34‐negative and PAS‐positive) in melanoma. (C) Kaplan–Meier survival curve demonstrating that high Rictor, AKT, MMP‐2 expression and VM positive are significantly related to poor prognosis (all P < 0.05).
Fig 3: Loss of Rictor in tubular cells aggravates renal inflammation after LPS administration.a Representative immunofluorescent staining images for Ly6b, CD3, and P65NF-κB among groups as indicated. White arrows indicate the NF-κB p65-positive tubular cells. Scale bar = 20 μm. b–d Quantitative determination of Ly6b+, CD3+, and P65NF-κB+ cells among groups as indicated. Each vertical bar represents the mean ± SEM (n = 3–6) analyzed by one way analysis of variance (ANOVA) followed by post hoc comparisons by Student–Newman–Keuls test. *p < 0.05 compared between different groups. e Real-time qRT-PCR analysis showing the mRNA abundance for TNF-α, Rantes, and IL-6 in Rictor+/+ and the knockout mice after LPS administration. Each vertical bar represents the mean ± SEM (n = 6) analyzed by students t test. *P < 0.05 vs. CDH16+-Rictor+/+ mice treated with LPS.
Fig 4: RICTOR was the target gene of miR-152. Note: A, The complementary binding sequence of RICTOR to miR-152 shown in the target gene prediction website; B, Dual-luciferase reporter gene assay verified the targeting relationship between RICTOR and miR-152.
Fig 5: LPS triggers Rictor/mammalian target of rapamycin complex 2 (mTORC2) signaling activation in tubular cells.a The graph showing the blood urea nitrogen (BUN) levels in CD1 mice at day 1 after treated with Vehicle or LPS. Each vertical bar represents the mean ± SEM (n = 7) analyzed by students t test. *P < 0.05 vs. vehicle mice. b Kidney histology as shown by periodic acid-Schiff (PAS) staining. Scale bar = 20 μm. c Western blotting assay showing the induction of p-Akt (Ser473) in the kidneys with LPS administration compared with those administrated with the vehicle. The numbers indicate the individual animal within each group. d Representation images showing the induction of p-Akt(Ser473) in the kidneys at day 1 after LPS. Scale bar = 20 μm. e NRK-52E cells were treated with LPS (500 ng/ml) for different time points as indicated. Western blotting analysis showing the induction and activation of Rictor/mTORC2 signaling and NF-κB pathway after LPS treatment in a time-dependent manner. f Representation images showing the immunostaining for Rictor and P65NF-κB in NRK-52E cells after LPS treatment. Scale bar = 5 μm.
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