PMC

Immunofluorescence of NKX2.1 and pro-surfactant protein (SP)-C in normal rat lung. A: double immunofluorescence for pro-SP-C (green) and NKX2.1 (red) shows that not all NKX2.1⁺ cells are pro-SP-C⁺. Negative controls are goat IgG (gIgG) and mouse IgG (mIgG). 4′,6-Diamidino-2-phenylindole (DAPI, blue) is the nuclear counterstain. Scale bar = 50 μm. B: confocal z-stack images demonstrate that some NKX2.1⁺ cells are pro-SP-C⁻ (arrows). Scale bar = 10 μm.

AT1 and AT2 cell markers in human lung tissue. A: not all NKX2.1⁺ cells colocalize with pro-SP-C in normal human lung (arrow, confocal z-stack image). DAPI (blue) was used to identify nuclei. Bar = 20 μm. B: ABCA3⁺ cells (>95%) coexpress pro-SP-C. Bar = 20 μm. C: many cells that express HOPX (red) also express the AT2 cell marker ABCA3 (green). Bar = 20 μm. D: most cells that express nuclear HOPX are also NKX2.1⁺. Bar = 20 μm.

Expression of AT1 and AT2 cell markers changes over time in culture during in vitro transdifferentiation. A: immunoblot with AT1 and AT2 cell markers shows HOPX is highly expressed from day 1 and increases only minimally in culture. AQP5 is expressed at very low levels on day 1 and increases over time. NKX2.1 decreases as cells transdifferentiate to the AT1-like phenotype; n = 3. B: diagram of proposed sequence of temporal changes in AT1 and AT2 cell markers as AT2 cells transdifferentiate toward AT1-like cells. Pro-SP-C expression drops abruptly as cells begin to transdifferentiate while NKX2.1 expression declines more slowly as cells progress though the intermediate (transitional) cell time period.

Immunofluorescence for alveolar type 1 (AT1) and 2 (AT2) cell markers in normal rat lung. A: left, immunofluorescence for homeodomain only protein x (HOPX, red) shows both nuclear and cytoplasm/membrane staining. Middle, magnified views of the rectangles shown on left. Right, negative control is rIgG. DAPI (blue) is the nuclear counterstain. Bar = 20 μm. B: double immunofluorescence shows HOPX (red) does not colocalize with pro-SP-C (green). Bar = 20 μm. C: double immunofluorescence shows nuclear HOPX (red) colocalizing with NKX2.1 (green). Bar = 20 μm. D: negative controls for C are rIgG and mIgG. E: triple immunofluorescence shows that NKX2.1 (light blue) colocalizes with either nuclear HOPX (red) or pro-SP-C (green), but not both. High magnification of rectangle is shown on bottom with individual channels. Bar = 20 μm. F: immunofluorescent localization of NKX2.1 (red) and AQP5 (green). Bar = 20 μm. Some cells are seen that colocalize both markers (arrows). G: immunofluorescent localization of HOPX (red) and AQP5 (green). Bar = 20 μm. Although most cells coexpress both markers, some do not (arrows).