(A) Schematic method of the matrix sandwich protocol. Defined ECM proteins were tested as coating matrix (blue) and overlay matrix (red). (B) Fluorescence images of DF19-9-11T iPSCs growing on the …
Scale bar is 100 µm.
The iPSCs were grown for 5 days on collagen IV coated surface and did not form confluent monolayer.
N≥3 biological replicates. *p<0.05, one-way ANOVA with post-hoc Bonferroni and test.
(A) Phase contrast and fluorescence images of the matrix sandwich culture of DF19-9-11T iPSCs grown for 4 days and immunolabeled using anti-FN antibody, compared with the monolayer culture. Scale …
Images and quantitative analysis for Figure 2B.
Scale bar is 100 µm.
(A) qRT-PCR for gene expression of EMT markers at days 0–3 of cardiac differentiation. (B) qRT-PCR for gene expression of mesendoderm/mesoderm and cardiac transcription factors at days 0–5 of …
The cells were co-labeled with antibodies against Brachyury (BRY) and FN. Scale bar is 25 µm.
(A) Schematic of the inducible shRNA construct for FN1 knockdown. (B) Schematic method of FN knockdown at differentiation stages of days 0–1, 1–5, and 5–7 in the cardiac differentiation protocol. (C)…
Yellow, CDS where shRNA binds; Green, palindrome. Oligo-1 binds in exon 8; Oligo-2 binds in 3’ UTR.
(A) Doxycycline-induced mCherry expression in the hPSC culture of H1 FN1 knockdown clone. Scale bar is 200 µm. (B) Phase contrast and immunofluorescence images of FN expression in doxycycline hPSC …
(A) cTnT+ cells measured by flow cytometry at 15 days of differentiation of the FN1 knockdown clones (H1c8 and H1c34) showing the effect of FN knockdown at days 0–1 at different concentrations of …
(A) qRT-PCR for gene expression of EMT markers for the H1 FN knockdown clone in the cardiac differentiation time course of 0–48 hr at the no dox control, dox induction at day 0–1 and dox induction …
(A) Schematic for testing monoclonal antibodies to block integrin β1 (P5D2), α5 (P1D6), αV (P3G8), and α4 (P4G9). (B) cTnT+ cells measured by flow cytometry at 15 days differentiation when …
(A) cTnT+ cells measured by flow cytometry at 15 days differentiation when anti-human integrin β1 antibody was added on day –2 or day 0 at 3 µg/ml in the matrix sandwich protocol. (B) cTnT+ cells …
(A) Schematic to test inhibition of ILK by small molecule inhibitor Cpd22 in the matrix sandwich protocol. (B) cTnT+ cells measured by flow cytometry at 15 days differentiation when Cpd22 was added …
The % of cTnT+ cells in each group was normalized to the control group to combine replicates in multiple differentiation. N=3 biological replicates. Error bars represent SEM. *p<0.05, one-way ANOVA …
(A, D) Representative flow cytometry plots of cells collected at 18 hr of differentiation, labeled by an antibody recognizing AnnexinV and stained by propidium iodide (PI) in the Matrix Sandwich …
(A) Representative plots of flow cytometry of the cells collected at 18 hr of differentiation in absence or presence of cpd22 at concentrations of 1, 3, and 10 µM. (B, C) Average of total apoptotic …
(A) Representative western blot analysis of pAKT(Ser473) and total AKT as well as total protein staining from day 0 (Time 0) to day 1 (24 hr) of the matrix sandwich protocol with or without cpd22 …
Raw unedited gels and blots and images with the uncropped gels and blots for all three experiements performed with the relevant bands labelled.
Tables of primary antibodies and qRT-PCR primers.
(a) Primary antibodies used in immunocytochemistry (ICC) and flow cytometry (FC). (b) Primers for quantitative RT-PCR. (c) Primary antibodies used for immunoblotting.