Fig 2: Overexpression of TRIM44 accelerates human esophageal cancer (HEC) cell migration and invasion. A, Relative TRIIM44 protein levels in HEC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as a loading control; B,C, efficiency of TRIM44 overexpression and down-regulation in HEC cells; D,E, Transwell assays were used to measure the effects of TRIM44 overexpression and down-regulation on migration and invasion; F,G, wound-healing assays were used to evaluate the migration of KYSE510 and KYSE140 cells. (*P < .05, **P < .01, and ***P < .001)

Fig 3: Increased TRIM44 expression in human esophageal cancer (HEC) patients is associated with poor prognosis. A, Representative photos of TRIM44 staining in 100 paired HEC tissues and matched adjacent normal tissue. The top figures: adjacent non-tumor tissues; and the bottom figures: different staining intensities in HEC tumors; B, analysis of the integrated optical density (IOD) value of TRIM44 immunostaining in 100 paired HEC tissues adjacent normal tissue; C,D, high TRIM44 correlated with poor tumor differentiation (P = .001) and overall survival (OS) was also found; E,F, high TRIM44 correlated with high TNM stage (P = 6.46E-05) and OS was also found; G, Kaplan-Meier plots showing the OS associated with TRIM44 expression in gastric cancer. In red: patients with high TRIM44 expression and in black: patients with low TRIM44 expression (data retrieved from Kaplan-Meier Plotter); H, overexpression of TRIM44 correlated with poor tumor differentiation and higher TNM stage (TRIM44high: high expression of TRIM44, TRIM44low: low expression of TRIM44; *P < .05, **P < .01, and ***P < .001)

Fig 4: Expression of Trim44 in myocardium under physiological and pathological conditions. (A,B) Expression of TRIM44 in heart tissues from patients with hypertrophic cardiomyopathy (HCM) (n=6) and normal controls (n=3), detected using western blotting (A) and quantitatively analyzed using GAPDH for normalization (B). (C,D) Expression of Trim44 in heart tissues from rat models of cardiac hypertrophy/heart failure induced by isoprenaline (ISO) or angiotensin (ANG II) treatment, detected using western blotting (C) and quantitatively analyzed using ß-actin for normalization (D) (n=4 rats per group). (E) Immunohistochemical analysis of Trim44 expression in paraffin sections of myocardial tissue from wild-type (WT) rats treated with ISO or saline and Trim44 KO rats at 3.5 months of age (scale bars: 20 µm). (F,G) Expression of Trim44 in myocardium from control rats at 1, 2, 3, 5, 7, 12 and 18 months of age, detected using western blotting (F) and quantitatively analyzed using ß-actin for normalization (G) (n=3 rats per time point). (H) Trim44 and cardiac troponin T (cTnT) immunofluorescence in paraffin sections of heart tissues from control and KO rats at 1 month of age (scale bars: 10 µm). **P<0.01, ***P<0.001 versus normal or saline group (unpaired two-tailed Student's t-test).

Fig 5: Trim44 KO attenuates ISO-induced cardiac remodeling through inhibition of the AKT/mTOR pathway. (A-E) Expression of phosphorylated (p-) and total AKT, P70S6K, GSK3ß and mTOR in heart tissue from control-saline, KO-saline, control-ISO and KO-ISO groups 2 weeks after cessation of ISO treatment, detected using western blotting (A) and quantitatively analyzed using the respective total protein for normalization (B-E) (n=4 rats per group). (F-J) Expression of phosphorylated and total AKT, P70S6K, GSK3ß and mTOR in H9c2 cells from the empty-vector group and Trim44 overexpression group (referred as Trim44-OV) with or without inhibitor treatment (PI3K/AKT inhibitor, LY294002; 25 µM), detected using western blotting (F) and quantitatively analyzed using the respective total protein for normalization (G-J) (n=4 replicates per group). (K) WGA immunofluorescence (scale bars: 40 µm). (L) Quantitative analysis of cross-sectional area of H9c2 cells for four groups (n=3-4 replicates per group, 54 cells per group). (M) Quantitative analysis of Nppb expression through real-time PCR for four groups, using Gapdh for normalization (n=3 replicates per group). *P<0.05, **P<0.01, ***P<0.001, ###P<0.001 versus respective control group (one-way ANOVA). NS, not significant.