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. 2023 Jun 14;11:1173149. doi: 10.3389/fbioe.2023.1173149

FIGURE 4.

FIGURE 4

Cell and genetic analysis of progenitor differentiation markers on day 15 between control and two experimental groups (static and dynamic). (A) Lung progenitor protocol. (B) qRT-PCR analysis of the expression of selected markers on day 15. qRT-PCR of lung progenitor markers. Bars show the mean fold change on day 15; error bars show one standard deviation; n = 3 biological replicates; data normalized to day 0 (2−ΔΔCT). p-values obtained by one-way ANOVA ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05. (C) TEM of dynamic cultured cells on day 15. (I) Loosely organized membranes of immature lamellar bodies (red arrows). Scale bars, 5 µm. II) MV = surface of the epithelial layer or surface microvilli, MVB = multivesicular bodies, and red arrows show electron-dense lamellar bodies. Scale bar, 1 μm; III) LB = lamellar bodies; G = glycogen particles trapped inside immature lamellar bodies. Scale bar, 2 µm. IV) Lipid droplets (LP) and microvilli (MV) and immature lamellar bodies (red arrow). Scale bars, 2 µm. (D) TEM of static cultured cells on day 15. TEM images: (I) MV = microvilli. Scale bars, 5 μm; II) LB = an immature lamellar body, MV = microvilli, small red arrow indicates extracellular space containing small debris, typical of an apoptotic cell. Scale bars, 2 μm; III) LB = an immature lamellar body; IV) Cells formed aggregates inside the beads and showed heterogeneity in cell morphology. The red arrow points to a typical apoptotic cell. Scale bar, 10 μm; long arrow points to the surface of the epithelial layer or the surface of cells (no microvilli). The small arrow points to the extracellular space showing small debris, typical of an apoptotic cell. Scale bars, 10 µm. (E) Immunohistochemical analysis of selected markers on day 15. Representative immunohistochemistry images with antibodies against pro-SFTPB (I and II), pro-SFTPC (III and IV) and Nkx2.1 (V and VI) on day 15 in static and dynamic cultures. Scale bar, 20 µm. RI, rock inhibitor; ANOVA, analysis of variance; TEM, transmission electron microscopy; pro-SFTPC, surfactant protein C; pro-SFTPB, surfactant protein (B).