Fig 1: Rheb1 is dispensable for high glucose–induced mTORC1 activation, and ablation of Rheb1 aggravates podocyte injury in streptozotocin-induced diabetic mice.(A) Western blot assay showing the abundance of Rheb1 in cultured podocytes after high glucose treatment at different times. (B) GTP loading assay showing the induction of GTP-Rheb1 after high glucose treatment. (C) Western blot assay showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, and p-mTOR in high glucose–cultured podocytes transfected with scramble or Rheb1 siRNA for 24 hours. (D and E) The cultured podocytes were pretransfected with scramble or Rheb1 siRNA for 24 hours, or treated with rapamycin for 30 minutes, followed by high glucose treatment for 24 hours. Propidium iodide (PI) staining (D) and quantitative analysis (E) of dead cells among different groups. Data are presented as the percentage of PI-staining-positive cells. #P < 0.05 vs. scramble control cells, n = 3; *P < 0.05, $P < 0.05 vs. high glucose–treated podocytes, n = 3. Scale bar: 20 μm. (F) UACR in Podo-Rheb1+/+ and Podo-Rheb1–/– mice at 6 months after STZ-induced DM. *P < 0.05 vs. Podo-Rheb1+/+ mice, n = 6–7. (G) Representative PAS staining and immunofluorescence staining for diabetic kidney injury, WT1, and nephrin among different groups. Scale bar: 50 μm. (H–J) Quantitative analyses of injury score, glomerular area, and WT1-positive podocytes per glomerulus among different groups. #P < 0.05 vs. vehicle control mice; *P < 0.05 vs. Podo-Rheb1+/+ mice with STZ injection, n = 3–7. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test (B and E). Comparison between the groups was performed using the 2-tailed Student’s t test (paired t test) (F). Data in H–J were analyzed with 2-way ANOVA with Tukey’s post hoc test.
Fig 2: mTOR signaling pathway was activated in cardiac tissues in chronic alcohol intake rats.(A) Western blotting images of Rheb, S6K2 and Rictor in heart tissue samples from model rats after 20 weeks ethanol consumption and the control rats. (B) Relative band intensities of Rheb, S6K2 and Rictor, in heart tissue samples. (C) Western blotting images of p-mTOR (ser 2448) in heart tissue samples. (D) Relative band intensities of p-mTOR (ser 2448). (E) Real-time quantitative PCR for Rheb, S6K2, and Rictor in heart tissue samples (n = 6 per group). Data are presented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01.
Fig 3: Activation of mTORC1 in KCs aggravates Con-A induced hepatitis.a The strategy for inducing macrophage Tsc1 deletion in Csf1r-Cre+/−, Tsc1fl/fl mice and Con-A administration. b The ALT levels in serum of mice exposed to Con-A for 8 h. Veh. n = 3, Con-a n = 7. c Representative HE and TUNEL- stained mouse livers. Scale bar = 200 μm as indicated in figures. d Left, representative immunofluorescent staining images for F4/80 with p-S6 (white arrows). Scale bar = 50 μm. Middle, quantitative determination of F4/80+ cells among groups as indicated, n = 3. Right, quantitative determination of F4/80+ and p-S6+ cells among groups as indicated, n = 3. e Left, representative immunofluorescent staining images for F4/80 with Ki67 (white arrows). Scale bar = 50 μm. Right, quantitative determination of F4/80+ and Ki67+ cells among groups as indicated. Veh. n = 3, Con-A n = 4. f Left, representative immunofluorescent staining images for F4/80 with CCP3 (white arrows). Scale bar = 50 μm. Right, quantitative determination of F4/80+ and CCP3+ cells among groups as indicated. Veh. n = 3, Con-A n = 4. g The strategy for inducing macrophage Rheb deletion in Csf1r-Cre+/−, Rhebfl/fl mice and Con-A administration. h The ALT levels in serum. Veh. n = 3, Con-A n = 7. i Representative HE and TUNEL- stained mouse livers. Scale bar = 200 μm or 100 μm as indicated in figures. j Left, representative immunofluorescent staining images for F4/80 with p-S6 (white arrows) and quantification. Scale bar = 50 μm. n = 4. k Left, representative immunofluorescent staining images for F4/80 with Ki67 (white arrows). Scale bar = 50 μm. Right, quantitative determination of F4/80+ and Ki67+ cells among groups as indicated, n = 4. l Left, representative immunofluorescent staining images for F4/80 with CCP3 (white arrows). Scale bar = 50 μm. Right, quantitative determination of F4/80+ and CCP3+ cells among groups as indicated, n = 4. *p < 0.05.
Fig 4: miR-365-3p directly targeted Rheb in NRK-52E cells and overexpression of Rheb reversed the protective effect of miR-365-3p. (A) Predicted miR-365-3p target sequence in Rheb-3′ UTRs. Target sequences of Rheb-3′ UTRs were mutated. (B) Luciferase assay of 293T cells transfected with Rheb-3′ UTR-WT or Rheb-3′ UTR-Mut reporter together with mimics NC or miR-365-3p mimics (n = 3). (C) Western blot analyzed Rheb protein levels in untreated NRK-52E cells (Control) and NRK-52E cells treated with mimics NC or miR-365-3p mimics, and quantification of Rheb protein level, n = 3 per group, GAPDH as an internal control. (D) Western blot analyzed Rheb protein levels in NRK-52E cells treated with control (PBS), mVNS-Exo or mVNS-Exo + miR-365-3p inhibitor, and quantification of Rheb protein level, n = 3 per group, GAPDH as an internal control. (E) NRK-52E cells were treated with Ad-Rheb or Ad-ctrl. Immunoblot analysis and quantification of p62 protein level, LC3II/LC3I ratio in NRK-52E cells, n = 3 per group, GAPDH as an internal control. (F) Representative images and semi-quantitative analysis of autophagosomes (yellow dots in the merged image) and autolysosomes (red dots in the merged image) in NRK-52E cells, n = 3 per group. Scale bar: 15 μm. ****p < 0.0001 versus CM group; ##p < 0.01 versus CM + miR-365-3p mimics group. &&p < 0.01 versus CM + miR-365-3p mimics + Ad-Rheb group. (G) NRK-52E cells were treated with miR-365-3p mimics, miR-365-3p mimics + Ad-Rheb or Rapamycin. Immunoblot analysis and quantification of p62 protein level, LC3II/LC3I ratio and p-mTOR/mTOR ratio in NRK-52E cells, n = 3 per group, GAPDH as an internal control. Data are mean ± SEM. *indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001; NS means not significant between groups. All p values were obtained by one-way ANOVA
Fig 5: Effects of miR-155-5p upregulation on the mTOR signaling pathway.(A) The expression of Rheb, S6K2 and Rictor in rats heart tissue at 15days post-injection was detected by western blot (n = 8 per group). (B) Relative band intensity of Rheb, S6K2 and Rictor in ACM heart tissue (n = 8 per group). (C) Western blot of p-mTOR (ser 2448) in ACM heart tissue (n = 8 per group) transfected with AAV-NC and AAV-miR-155 (n = 8 per group). (D) Relative band intensity of p-mTOR (ser 2448) normalized to mTOR. (E) Real-time quantitative PCR for Rheb, S6K2, and Rictor in heart tissue samples (n = 8 per group). Data are presented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01.
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