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. 2021 Nov 4;9:737840. doi: 10.3389/fcell.2021.737840

FIGURE 2.

FIGURE 2

(A) From left to right: confocal images of immunostained 2D monolayer cultured hiPSC-CM, 3D-cultured hiPSC-CM tissue, 2D-cultured hiPSC-CM seeded on Matrigel mattresses, and treated with T3+Dexamethasone and cryosections of adult human heart tissue. Adult heart sections were counterstained with wheat germ agglutinin (WGA) for membrane, shown in magenta. hiPSC-CM were counterstained with cardiac troponin T (cTnT) or α-actinin, shown in magenta. Nuclei were labeled with DAPI in blue. The DAPC components are in green. Results were replicated in three independent hiPSC-CM differentiations. Scale bar, 10 μm. (B) (a) Immunoblot of dystrophin (Dys) and dystroglycan (DG). (b) Immunoblot of sarcoglycans (SG) and Deglycosylation tests (+PNGase treatment) in adult cardiac homogenates and in 3D-cultured hiPSC-CM. Results were replicated in seven independent hiPSC-CM differentiations for α-SG, δ-SG, and γ-SG; 10 independent differentiations for β-SG and dystrophin and 5 independent differentiations for dystroglycan (DG). (c) Immunoblot for sarcoglycans from 3D-cultured hiPSC-CM treated for 8 h with MG-132 (10 μM). The proteasome inhibition efficiency was confirmed by immunoblotting of lysates with an anti-ubiquitin antibody (right). Results are from four 3D constructs prepared from one hiPSC-CM differentiation. Straight lines separate adult vs. hiPSC-CM, and dotted lines separate control vs. treatment (PNGase or MG132), from the same blot. The expected molecular weight for glycosylated and deglycosylated forms of sarcoglycans are indicated in the figure. The β-SG band at 50 kDa was considered as non-specific as its molecular weight did not decrease with deglycosylation (a similar band was also seen in β-SG-null mouse heart—Supplementary Figure 2B). (C) mRNA detection by reverse transcription PCR of expression of sarcoglycans in 2D and 3D-cultured hiPSC-CM and in adult human heart lysates. Results were replicated in three independent hiPSC-CM differentiations. (D) RT-qPCR of sarcoglycans in 3D-cultured hiPSC-CM and adult human heart lysates. Values are expressed as 2–ΔΔCT normalized as a fold difference to adult.