Fig 2: Functional interrogation of TTN c.15286T>C, p.C5096R HEK293T cells reveals lack of contact inhibition phenotype. a Wildtype and mutant cells were serially counted at 24-h interval for up to 144 h after seeding. Trypan blue stain was used to count dead cells and assess viability. Experiments were performed as four biological replicates, and counted at least in triplicates at each time point. Data represent pooled mean values ± SEM. **p < 0.001, *p < 0.05 (two-sided Student’s t test comparing each mutant cell line to wildtype). b Western blot and densitometric analysis of some downstream readouts of the PTEN signaling pathway. Blots were derived from the same experiment and representative of three independent biological replicates, with protein samples from each cell line (genotype) processed in parallel within each experiment. Densitometric data represent pooled mean values of the replicate experiments ± SEM. **p < 0.005, *p < 0.05 (two-sided Student’s t test comparing each mutant cell line to wildtype). c Western blot and densitometric analysis of total and phosphorylated FAK protein. Blots were derived from the same experiment and representative of two biological replicates, with protein samples from each cell line (genotype) processed in parallel within each experiment. p-FAK Y397, autophosphorylation site at tyrosine residue 397; p-FAK Y576/577, catalytic domain phosphorylation at tyrosine residues 576/577; p-FAK Y925, carboxy-terminal region phosphorylation at tyrosine residue 925. Densitometric data represent pooled mean values of the replicate experiments ± SEM. *p < 0.05 (two-sided Student’s t test comparing each mutant cell line to wildtype). Both Western blot experiments were performed on lysates from cells harvested at the 144 h timepoint (Day 6). Non-phosphorylated proteins are normalized to GAPDH (loading control), whereas phosphorylated proteins are normalized to corresponding total protein levels after normalization of both proteins to GAPDH (ratio of the ratios). Fold changes are calculated relative to wildtype (set to 1). Molecular weights of proteins in kilo Daltons (kDa) are indicated on the left side of each blot

Fig 3: DCYTB expression inhibits adhesion to fibronectin. a Phosphorylation of FAK and paxillin was evaluated by Western blot in MCF7 cells expressing doxycycline-inducible DCYTB or control empty vector (EV). Quantification of staining intensity was performed using ImageJ. b Adhesion of MCF7 cells expressing DCYTB or control empty vector (EV) to fibronectin. c Phosphorylation of FAK and paxillin in SKBR3 cells expressing doxycycline-inducible DCYTB or control empty vector (EV). d Adhesion of SKBR3 cells expressing DYCTB or control empty vector (EV) to fibronectin. Graphs represent means and standard deviation of 16 replicates and are representative of three independent experiments. DCYTB duodenal cytochrome b

Fig 4: Serpina1e is required for the inhibitory effects of AKG on hepatic gluconeogenesis.(A and B) Principal coordinate analysis plot (A) and volcano plot of AKG-induced transcriptome signature (B) (n = 3). (C) mRNA expression of serpina1e in si-serpina1e–treated primary hepatocyte (n = 6). (D to F) Immunoblots (D) and quantification (E), and enzyme activity (F) of PEPCK, G6Pase, and FBP in primary hepatocyte (PA treatment) (n = 4 to 8). (G) mRNA expression of serpina1e in the liver (n = 8). (H and I) Immunoblots (H) and quantification (I) of p-FAK and p-Akt protein expression in the liver (n = 3). (J) mRNA expression of serpina1e in Alb-serpina1e-/- mice liver (n = 8). (K to M) Blood glucose (K), PTT (L), and ITT (M) in Alb-serpina1e-/- mice (n = 8). (N to P) Immunoblots (N) and quantification (O), and enzyme activity (P) of PEPCK, G6Pase, and FBP in the liver (n = 4 to 8). (Q) Serum insulin level (n = 8). (R) mRNA expression of JMJD3, LSD1, and UTX in AKG-treated primary hepatocyte (PA treatment) (n = 8). (S and T) mRNA (S) and protein (T) expression of JMJD3 in si-JMJD3–treated primary hepatocyte (n = 3 to 6). (U) Chromatin immunoprecipitation analysis of H3K27me3 in promoter of serpina1e in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 8). (V) mRNA expression of serpina1e in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 8). (W to Y) Immunoblots (W) and quantification (X), and enzyme activity (Y) of PEPCK, G6Pase, and FBP in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 4 to 8). Results are presented as means ± SEM. In (E) to (G), (O) to (Q), (U), (X), and (Y), different letters between bars indicate P = 0.05 by one-way ANOVA followed by post hoc Tukey’s tests. (C, I, J, R to T, and V) *P = 0.05 by nonpaired Student’s t test. (K to M) *P = 0.05, **P = 0.01 by two-way ANOVA followed by post hoc Bonferroni tests.

Fig 5: Effects of the administration of irisin on bone in nephrectomy (Nx) mice. (A) Cortical bone mineral density (CtBMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), and trabecular bone mineral density (TbBMD) at the femur of mice were assessed by QCT 8 weeks after the last Nx or sham surgery. (B) Total RNA was extracted from the femurs of mice 8 weeks after the last Nx or sham surgery. A real-time PCR analysis of Runx2, osterix, ALP, osteocalcin (OCN), and 18S rRNA was performed. Data are expressed relative to the levels of 18S rRNA. (C) Immunohistochemistry of ALP in the femurs of mice 8 weeks after the last Nx or sham surgery. The number of ALP-positive cells per 1 mm2 of the cortical bone surface was counted. Red colour indicates ALP-positive signals. Scale bars indicate 25 µm. (D) TRAP staining was performed on the femurs of mice 8 weeks after the last Nx or sham surgery. The number of TRAP-positive multinucleated cells (MNCs) per 1 mm2 of the cortical bone surface was counted. Scale bars indicate 25 µm. (E) Total proteins were extracted from the femurs of mice 8 weeks after the last Nx or sham surgery. Western blot analyses of phosphorylated Akt (pAkt) at Thr308, Akt, phosphorylated focal adhesion kinase (FAK) at Thr389, FAK, and GAPDH were performed. Images represent experiments performed independently at least four times. Data represent the mean ± standard error of the mean. n = 8 mice (A–D) and 4 mice (E) in each group. Cont, control. *P < 0.05 and **P < 0.01.