(A) Cellular compartments in which proSFTPB processing into mature SFTPB occurs. MVB, multivesicular body.
(B) Western blot of a time course of alveolosphere differentiation (days 0, 16, 22, 29, and 36) and 6-day DCI-cultured primary week 21 human fetal distal lung (HFL DCI-D6) as a positive control. Top: use of an anti-NFLANK antibody that binds to the N-pro region of pro-SFTPB intermediates. Bottom: use of an anti-mature SFTPB antibody (PT3) that binds all SFTPB forms, including the 8-kD mature form.
(C) Dissociation protocol for isolation of intracellular and extracellular components of alveolospheres. Both absolute (nanomoles of lipid/micrograms of DNA in cell samples) and relative (nanomoles of lipid/nanomoles of total diacyl phosphatidylcholine) are shown for both surfactant-specific 32:0 dipalmitoyl phos-phatidylcholine (DPPC) and nonspecific 34:1 phosphatidylcholine (PC). The surfactant index was calculated as the sum of the surfactant-specific 30 and 32 PCs divided by the sum of non-surfactant-specific 36 PCs. Bars represent mean ± SD for each analysis, n = 3 biological replicates separated on day 15; *p % 0.05, **p % 0.01, ***p % 0.001, ****p % 0.0001 by unpaired, two-tailed Student’s t test.