Fig 2: TAZ induces the expression of Rheb and Rhebl1 to promote the initiation of the translation of Tfam mRNA.a RNA was isolated from the gastrocnemius muscle of wild-type (WT) and muscle-specific TAZ knockout (mKO) mice, and the transcript levels of Rheb and Rhebl1 were assessed using quantitative reverse transcription (qRT)-PCR; n = 6 (left), n = 7 for WT mice, and n = 6 for mKO mice (right) (Rhebl1; *p = 0.0426, Rheb; ***p = 0.0002). b Protein derived from the gastrocnemius muscle of WT and mKO mice was analysed via immunoblotting to determine the Rheb and Rhebl1 protein levels. Vinculin was used as the loading control. c The expression of Rheb and Rhebl1 was assessed using qRT-PCR in WT, TAZ knockout (KO), and TAZ-rescued (T) mouse embryonic fibroblasts (MEF). The experiment was performed in triplicate (Rhebl1; ***p = 0.0001 for WT vs. KO, ****p < 0.0001 for KO vs. T, Rheb; ***p = 0.0007 for WT vs. KO, **p < 0.003 for KO vs. T). d Protein from WT, KO, and T MEF was analysed via immunoblotting to detect TAZ, Rheb, and Rhebl1. Vinculin was used as the loading control. e RNA was isolated from control (Con) and C2C12 myotubes with TAZ knockdown (KD). Rhebl1 and Rheb expression was assessed using qRT-PCR. The experiment was performed in triplicate (Rhebl1; *p = 0.0105, Rheb; *p = 0.0171). f Protein from the Con and TAZ KD C2C12 myotubes was analysed via immunoblotting to detect the Rhebl1 and Rheb protein levels. Alpha-tubulin was used as the loading control. g Recruitment of TAZ (left) or TEAD4 (right) to the TAZ binding element (TBE) of the Rhebl1 enhancer was verified using chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR). Sheared chromatin from the gastrocnemius muscle of WT mice was ChIPed with the anti-TAZ or anti-TEAD4 antibody. ChIPed DNA was analysed via qPCR using primers spanning the TBE region. Immunoglobulin G (IgG) was used as the ChIP control. The experiment was performed in triplicate (TAZ; *p = 0.0183, Tead4; ***p < 0.0001). h TEAD4 was depleted by two different siRNAs in FLAG-tagged TAZ (F-TAZ)-overexpressing C2C12 myoblasts, and ChIP was performed using an anti-FLAG antibody. ChIPed DNA was assessed via qPCR using a primer set covering the TBE region. Empty vector-transduced cells were used as ChIP controls. The experiment was performed in triplicate (Con vs. F-TAZ + Scrambled; **p = 0.0011, F-TAZ + Scrambled vs. F-TAZ + Tead4 si #1; *p = 0.0112, F-TAZ + Scrambled vs. F-TAZ + Tead4 si #2; **p = 0.0025). i Schematic description of Rhebl1 cis-regulatory element regulated by TAZ-TEAD4. The transcriptional activity of the Rhebl1 TBE was confirmed using a luciferase reporter gene assay. Approximately 500 bp of the genomic region covering the Rhebl1 TBE were cloned into a pGL3 basic luciferase reporter plasmid together with the promoter region of Rhebl1 (Rhebl1 TBE-Luc). Rhebl1 TBE-Luc was transfected into HEK293T cells with TAZ and/or TEAD4, and a reporter gene assay was performed 24 h after transfection. The experiment was performed in triplicate (Rhebl1 TBE vs. Rhebl1 TBE + TAZ; **p = 0.0033, Rhebl1 TBE + TAZ vs. Rhebl1 TBE + TAZ + Tead4; ***p = 0.0007). j Con and HEK293T cells with TAZ KD were transfected with Rhebl1 TBE-Luc. A luciferase reporter assay was performed 24 h after transfection. The experiment was performed in triplicate (***p = 0.0002). For a, b, g 8 to 10-week-old mice were used. Data are presented as mean ± SEM for (a), and mean ± SD for (c, e, g–j). Statistical significance was analysed via two-sided t-test for (a, e, g) or one-way ANOVA with Tukey’s multiple comparison test for (c, h, i). For (j) two-way ANOVA with Sidak’s multiple comparison test was used. Representative data was shown and experiments were performed at least twice with similar results for (b, d, f). Source data are provided as a Source data file.

Fig 3: siRNA knockdown of the DDIT4 antiproliferative responses to 1,25(OH)2 D3RMCs were transfected with negative control siRNA orDDIT4-specific siRNA in the presence or absence of 1,25(OH) 2 D3 . (A) The proliferative activity of the transfected RMCs was detected using the cell counting kit-8 assay. ap < 0.05, DDIT4 siRNA vs. control (n = 3). (B) Cell-cycle distribution and size of the transfected RMCs analyzed using flow cytometry. (C) DDIT4, TSC1/TSC2, Rheb, mTOR, 4E-BP1 and p70S6K protein expression levels were detected by Western blot. *p < 0.05, DDIT4 siRNA vs. control; #p < 0.05, DDIT4 siRNA+VD vs. DDIT4 siRNA (n = 3).

Fig 4: Effect of microRNA‐155 (miR‐155) inhibition and overexpression on the endothelial cell junction proteins. Immunofluorescence microscopy (A) and Western blot analysis (B) were performed to detect the cellular distribution and expression levels of cell junction proteins in oxygen‐glucose deprivation (OGD)/control inhibitor (OGD/IC), specific miR‐155 inhibitor (OGD/I), and mimic (OGD/M) cells. A, Immunofluorescence staining for occludin, claudin‐5, and zonula occludens protein‐1 (ZO‐1; red). Confocal microscopy images were acquired using a Zeiss LSM800 confocal microscope. Bar: 10 μm. B, Representative immunoblots demonstrate the levels of occludin, claudin‐5, ZO‐1, and vascular endothelial (VE)–cadherin. In addition to cell junction proteins, Western blots were performed to assess expression of miR‐155 direct target Rheb and RhoA bands. C, Graph demonstrates quantification of Western blot data obtained in OGD/I (black squares) and OGD/M (grey triangles) samples. Optical density of the protein bands was measured using ImageJ software, normalized to GAPDH density in every sample, and expressed as a percentage of the optical density calculated in the appropriate control OGD/IC and mimic control (OGD/MC) samples, respectively. n=4 (for OGD/IC and OGD/MC groups) and 6 (for OGD/I and OGD/M groups) independent experiments. Mann–Whitney (Wilcoxon) test was used to compare the relative protein levels between OGD/I and OGD/IC groups (P=0.029). Error bars: SEM; *P<0.05.

Fig 5: RV infection downregulates let-7g expression. (A) HT29 cells were mock treated or infected with RV for indicated time points. Whole cell lysates were prepared, followed by western blot analysis using specific antibodies against Rheb-GTP, TSC1, TSC2, and RV-VP6. The blot was reprobed with an antibody to GAPDH for normalization. (B) In silico analysis of the TSC1 3′UTR revealed a single putative let-7g binding site. The let-7g target region of TSC1 (GenBank accession number NM_000368) is indicated. (C) HT29 cells were transfected with mimic let-7g (40 nM) (left panel) or pmR-ZsGreen1-pre-let7g (right panel) followed by western blot analysis using specific antibody against TSC1. Relative fold differences of the TSC1 level was analyzed after normalization with GAPDH from at least three independent experiments. (D) HT29 cells were mock treated or infected with RV-SA11 for indicated time points. Total RNA from mock- or virus-infected cells was extracted and expression of let-7g was analyzed by qRT-PCR and normalized to the expression of U6 snRNA. (E) RV-VP6 RNA level was measured and plotted as relative RNA level in comparison to mock-infected cells. (F) Cells were infected with UV inactivated RV-SA11. At 8hpi the total RNA was extracted and the relative expression of let-7g was quantified by qRT-PCR analysis. (G) VP6 RNA level was measured as a marker of viral infection in UV-inactivated RV-SA11 infected cells. Results are presented as the means and standard deviations from at least two independent experiments.