Fig 1: Characterization of the hiPSC-derived cardiomyocytes. a, b CHIR concentration-dependently increased the expression of BRACHYURY (a) and MESP1 (b) in differentiated cells at day 1 and day 3 (t test, *p < 0.05, **p < 0.01, and *** p < 0.001 versus 0 µM or day 1; n = 3). c The hiPSC-induced cardiomyocytes expressed cTNT and a-actinin indicated by IF assay in induced cardiomyocytes from day 21 differentiation. Scale bars, 100 µm (× 400). d Representative plots of flow cytometry displaying high yields of cardiomyocytes derived from hiPSC at day 21. Expression values of all PCR analyses were normalized to the housekeeping gene GAPDH. Data are presented as “mean ± SD’”
Fig 2: U0126 inhibited YB-1 phosphorylation and alleviated diabetic cardiomyopathy.A, animal experiment procedure. Eight-week-old male C57BL/6J mice were injected with 60 mg/kg STZ to induce diabetes mellitus, and controls received the same volume of sodium citrate. Diabetic mice were treated with 1 mg/kg U0126 or the same volume 6% DMSO in 26 weeks. All mice were sacrificed at the age of 34 weeks. B, blood glucose detected at 34 weeks in mice with different treatments. C, ratios of heart weight to body weight in mice with diverse treatments. D, representative histological images of transverse area of myocyte in mice with different treatments. For WGA staining, green: WGA, red: cTNT, and blue: DAPI. The scale bar represents 20 µm; for HE staining, the scale bar represents 50 µm. E and F, analyzed histological data showing the myocyte cell surface area in diabetic mice with or without U0126 treatment, relative to controls. G, representative echocardiography images of mice with various interventions in M mode. H and I, echocardiography analysis of EF and FS in mice with different interventions. J, representative immunoblotting images showing protein expression level of ANP, BNP, and ß-MHC in mice heart tissue lysates. K, representative immunoblotting images showing protein expression and phosphorylation levels of ERK, p90RSK, and YB-1 in mice treated with or without U0126, compared with controls. L, YB-1 immunoprecipitation in mice heart tissue lysates showing the impact of U0126 treatment on K48-linked ubiquitination of YB-1. M, interaction between YB-1 and OTUB1 detected by immunoprecipitation of YB-1 and OTUB1, respectively, in different group of mice heart tissue lysates. For all groups, n = 8. Data are expressed as mean ± SD. **p < 0.01; ns, One-way ANOVA, Bonferroni comparison test. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; cTNT, cardiac troponin T; DAPI, 4',6-diamidino-2-phenylindole; DM, diabetes mellitus; DMSO, dimethyl sulfoxide; EF, ejection fraction; ERK, extracellular signal-regulated kinase; FS, fraction shortening; K48, lysine 48; ß-MHC, ß-myosin heavy chain; ns, not significant; OTUB1, otubain-1; RSK, p90 ribosomal S6 kinase; STZ, streptozotocin; WGA, wheat germ agglutinin; YB-1, Y-box binding protein-1.
Fig 3: DNA damage and cellular apoptosis in cKO mice.(A) Schematic representation of the confiner device. (B) Percentage of nuclei positive for ?-H2AX in Lemd2fl/fl and cKO CMs isolated from P1 mice at baseline and after 20 µm compression for 1 hour. Three experimental replicates. n = 2–5 mice per genotype, more than 80 nuclei per replicate. ***P < 0.001; **P < 0.01; *P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. (C) Representative images of ?-H2AX and cTNT staining in CMs isolated from P1 Lemd2fl/fl and cKO mice at baseline and after 20 µm compression for 1 hour. Scale bar: 20 µm. (D) Percentage of TUNEL-positive nuclei in Lemd2fl/fl and cKO CMs isolated from P1 mice at baseline and after 20 µm compression for 1 hour. 2–3 replicates. n = 2–5 mice per genotype, more than 80 nuclei per replicate. **P < 0.01; *P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. (E) Representative images of TUNEL and cTnI staining in Lemd2fl/fl and cKO CMs isolated from P1 mice and compressed at 20 µm for 1 hour. Scale bar: 20 µm. (F) Nuclei area in CMs isolated from P1 Lemd2fl/fl and cKO mice at baseline and after 20 µm compression for 1 hour. n = 2 mice per genotype, 15–30 nuclei per genotype. *P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. (G) Nuclear solidity (area/convex area) in CMs isolated from P1 Lemd2fl/fl and cKO mice at baseline and after 20 µm compression for 1 hour. n = 2 mice per genotype, 15–30 nuclei per genotype. ***P < 0.001; *P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. (H) Representative images of lamin B1 and cTnI staining in Lemd2fl/fl and cKO CMs isolated from P1 mice and compressed at 20 µm for 1 hour. Scale bar: 5 µm.
Fig 4: Validation of the enriched differentiation of SANLC by the BMP4/PD/BMS (BPM). a–c Representative IF analysis showed that SANLCs expressed SAN-specific transcription factor, SHOX2, TBX3, and TBX18. Scale bars, 100 µm (× 400). d Representative flow cytometry analysis showed that BPM significantly increased the percentage of CTNT+/NKX2.5- cells as compared to the GiWi group (t test, ***p < 0.001 versus GiWi control; n = 5). e Representative flow cytometry analysis showed that BPM significantly increased the percentage of CTNT+/SHOX2+ cells as compared to the GiWi group (t test, ***p < 0.001 versus GiWi control; n = 5). Data are presented as “mean ± SD”
Fig 5: YB-1 phosphorylation was increased in diabetic mice heart.A, schematic diagram of animal experiment procedure. C57BL/6J male mice received 60 mg/kg STZ or sodium citrate for 5 days at the age of 8 weeks and sacrificed at 34 weeks. B, the ratios of heart weight to body weight in mice with diabetes mellitus compared with controls. C, blood glucose detected in different time points during the diabetic cardiomyopathy progression. D, representative images of transverse area of cardiomyocyte detected by WGA and HE staining. In WGA staining, green: WGA, red: cTNT, and blue: DAPI. The scale bar for WGA represents 20 µm, and the scale bar for HE represents 50 µm. E and F, histological analysis of WGA and HE staining. The areas of myocyte surface were calculated by Image-Pro Plus and standardized by control group. G, representative echocardiography images (M mode). H and I, echocardiography analysis of EF and FS. J and K, hemodynamic parameters measured by the Millar cardiac catheter system showing a reduction of cardiac function. L and M, representative immunoblotting images and analyzed scatter diagram showing the protein level of cardiac hypertrophy and dysfunction markers in heart tissue of mice with or without diabetes mellitus. N–P, immunoblotting images of phosphor-YB-1 (S102) and YB-1 protein expression. Analyzed data showed that the protein expression level of YB-1 was decreased, whereas its phosphorylation increased significantly. For all groups, n = 6. Data are expressed as mean ± SD. **p < 0.01, Student’s t test. cTNT, cardiac troponin T; DAPI, 4',6-diamidino-2-phenylindole; DM, diabetes mellitus; EF, ejection fraction; FS, fraction shortening; S102, serine102; STZ, streptozotocin; WGA, wheat germ agglutinin; YB-1, Y-box binding protein-1.
Supplier Page from Proteintech Group Inc for Cardiac Troponin T antibody