Additional file 1 of Stage-specific regulation of signalling pathways to differentiate pluripotent stem cells to cardiomyocytes with ventricular lineage
Creators: name: Satthenapalli, Ramakanth
name: Lee, Scott
name: Bellae Papannarao, Jayanthi
name: Hore, Timothy A.
name: Chakraborty, Akash
name: Jones, Peter P.
name: Lamberts, Regis R.
name: Katare, Rajesh
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1. Figure S1. A. Representative bright-field image of TNG-A mESCs aggregated in spherical colonies. B. Acolony of TNG-A mESCs showing the endogenous expression of pluripotent nanog protein due to the GFP transgene tagged to nanog. Scale bar=100μm. Figure S2. A&amp;C. Representative immunofluorescence images of mESCs on day 0 (A) and day 14 (time control, C) showing the expression of pluripotent markers (Oct4 and Sox2). Specificity of the antibody was confirmed by staining a independent samples with secondary controls only. B&amp;D. Quantitative scater plot bar graphs of RT-PCr analysis showing the expression of pluripotent markers gene Nanog, Oct-4 and Sox-2 in of mESCs on day 0 (B) and day 14 (time control, G). Mature mouse cardiomyocyte cell line HL-1 cells were used as negative controls. Figure S3. Representative confocal microscopy images showing the cells at different days of differentiation that are stained with secondary antibodies with the same labelling procedure without the primary antibody treatment, to rule out the non-specific binding of the secondary antibody. Figure S4. Quantitative scatter plot bar graphs of RT-PCR analysis showing the expression of cardiac differentiation genes in mESCs grown in differentiation medium with treatments (differentiated) and mESCs grown without any treatment (time control). Data were analyzed by unpaired t-test presented as Mean ± SEM. *P&lt;0.05. n=4 independent repeats. Figure S5. A. Representative image of Ca2+ activity in a single differentiated mESC-CM on day 14 (A). The cells did not show any propensity to spontaneously release the calcium when induced with different concentrations of Ca2+. B. Quantitative scatter plot bar graphs showing a significant response of differentiated cells to caffeine-induced Ca2+ release C. Quantitative scatter plot bar graphs of RT-PCR analysis showing the expression of RYR2 after 14 days of differentiation. Data were analyzed by unpaired t-test with Welch’s correction. **P&lt;0.01 and ****P&lt;0.0001 vs. differentiated cells. Data presented as Mean ± SEM. n=35 cells from three independent experiments in B n=4 independent experiments in C. Figure S6. Representative bright field microscopic images of m-ESC over 14days of cultivation by EB method. On day 2 (A) the EBs were generated and transferred to a petri dish. The colony was preserved and the EB was still in three-dimensional shape on day 3 (B), On day 4 (C) the EBs were transferred to gelatin coated plates and finally on day 5(D) the EB adhering to the plate was observed. From day 6 (E) the images were taken at a magnification of 40x to observe closer morphological changes. Cells looked more spherical and the cells were observed very close together. Over the period of day 7(F), 8 (G), 9(H), 10(I), 11(J), 12(K), 13(L), 14(M), the morphology of cells became more elongated and also the myotube formation was observed. Clear morphological differences can be seen comparing undifferentiated ESC (N) and differentiated cardiomyocytes on day14 (O). Scale bars are 500 μM for A-D, 200 μM for E-M and 00 μM for N&amp;O. Figure S7. A–B. Representative confocal microscopy images showing the expression of cardiac transcription factors Nkx2.5 (A) and Isl-1 (B) in differentiated and undifferentiated mESCs by EB method. C. Representative confocal microscopy images showing the expression of ventricular cardiomyocytes markers IRX4 and MLC2V following differentiation of mESCs by EB method in both +RAi and -RAi groups. D–E. Representative confocal microscopy images showing the expression of ventricular cardiomyocytes markers Mef2c (D) and Cx43(E) in differentiated and undifferentiated mESCs by EB method. F. Representative flowcytometry gating images showing the expression of left ventricular cardiomyocytes marker IRX4 and cTnt following differentiation of mESCs by EB method in both +RAi and -RAi groups. Figure S8. Uncropped western blots of Connexin43 (Cx43) and GAPDH represented in Figure 3C (A) and MLC2V and GAPDH represented in Figure 3D (B).