Fig 2: Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC toward Sinoatrial node-like cells. (A) BMS was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 5 μM BMS was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant, *denoting p<0.05, **denoting p<0.01, and ***denoting p<0.001.

Fig 3: SHOX2 promotes the migration and invasion of PCa cells(A–C) The impact of SHOX2 knockdown and overexpression on the migration of PCa cells was respectively shown using Transwell migration assays, with a scale bar of 200 μm.(D–F) The effects of SHOX2 knockdown and overexpression on PCa cell migration were evaluated using wound healing assays, with a scale bar of 200 μm. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 4: SAN marker expression in D21 cardiomyocytes. (A) Representative image of ICC staining of day 21 cardiomyocytes of R1, MAP3k7/TAK1-overexpressing, B1, and B1 DOX-treated EBs. EBs were dissociated into single-cell suspension and replated onto chamber slides. Then these cells were stained with GFP, DsRed or CT3 antibody to identified cardiomyocytes. HCN4 (R1 N=124, TAK1 N=233, B1 N=125, BIDOX N=110), Shox2 (R1 N=128, TAK1 N=175 B1 N=116, BIDOX N=146), and Cx43 (R1 N=108, TAK1 N=61, B1 N=88, BIDOX N=70) antibody were immunostained with red fluorophore, DAPI labelled nuclei blue. Scale bars: 30 µm. (B) The percentage of HCN4, Shox2 and Cx43 positive cells. Data represent means±s.e. of at least three independent experiments. Statistical significance was determined by unpaired two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5: Blocking TAK1 phosphorylation increases expression of SAN transcription factors in cardiomyocytes. (A) Beat rate data day 21 cardiomyocytes from R1, TAK1 constitutive, TAK1-inducible with DOX, and R1 with OXO. Statistical significance was determined by ANOVA. (B) Live cell imaging of day 21 cardiomyocytes from R1 and (C) R1 with OXO treatment, imaging fluorescence from the aMHC:GFP cardiac promoter reporter. (D) Quantification of Shox2 (control N=110, OXO N=80), Islet1 (control N=48, OXO N=44), and HCN4 (control N=118, OXO N=123) expression in cardiac cells as assessed by ICC, with and without OXO treatment. Data represents means±s.e. of at least three independent experiments. Statistical significance was determined by an unpaired two-tailed t-test. **P<0.01, ***P<0.001, ****P<0.0001.