DEV036798 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Normal expression of alveolar epithelial markers in TRIC-B-knockout neonatal lungs. (A) Immunostaining of propidium iodide-labelled lung tissues using antibodies to AQP5 (upper), mature SP-B (middle) and proSP-C (lower). Scale bar: 50 µm. (B) Statistical analysis of AQP5-positive type I cells and proSP-C-positive type II cells in the neonatal lung epithelia. The data represent the mean±s.e.m., and the numbers of neonates examined are shown in parentheses. (C) Primary alveolar epithelial cells in culture. In the phase-contrast images (left), arrowheads and arrows indicate type I and type II cells, respectively. In the fluorescence images (right), the cultured cells were stained with propidium iodide (red) and immunolabelled using antibodies to AQP5 or proSP-C (green). Scale bar: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Insufficient phospholipids in interstitial fractions from TRIC-B-knockout neonatal lungs. (A,B) LC-ESIMS/MS analysis of phospholipids in lung interstitial fractions from wild-type (A) and TRIC-B-knockout (B) neonates. The internal standard 17:0-LPC (IS, m/z 510.7) was added to the lipid extracts and used for normalising phospholipid contents analysed. Representative mass spectrum charts for phosphatidylcholine (PC) and lyso-phosphatidylcholine (LPC) species are shown. (C) Quantification of PC species in lung interstitial fractions. (D) Quantification of phosphatidylglycerol (PG) species in lung interstitial fractions. (E) Quantification of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) species in lung interstitial fractions. The data represent the mean±s.e.m., the numbers of neonates examined are shown in parentheses, and statistical differences between the genotypes are marked with asterisks (*P<0.05, **P<0.01 by Student’s t-test).

• Supplemental Figure S3 -

  Fig. S3. Absence of tubular myelin in TRIC-B-knockout lungs. In the alveolar space of wild-type neonatal lungs, tubular myelin formation was frequently seen (left). In the TRIC-B-knockout lungs, typical structures of tubular myelin could not be detected, but loose roll structures composed of a few lipid layers were often detected (right). Scale bar: 1 µm.

• Supplemental Figure S4 -

  Fig. S4. Abnormal Ca²⁺ responses in TRIC-B-knockout alveolar type II cells. (A) Ca²⁺ transients induced by the PAR4 agonist peptide (PAR4AP) in type II cells. (B) Impaired PAR4AP-evoked Ca²⁺ transients in TRIC-B-knockout type II cells. The data in A were statistically examined. (C) Ca²⁺ responses induced by thapsigargin (TG) in type II cells. (D) Ca²⁺ responses induced by cyclopiazonic acid (CPA) in type II cells. (E,F) Ca²⁺-overloaded stores and normal SOCE responses in TRIC-B-knockout type II cells. The data in C and D were statistically examined. The data represent the mean±s.e.m., the numbers of cells examined are shown in parentheses, and statistical differences between the genotypes are marked with asterisks (**P<0.01 by Student’s t-test).

• Supplemental Figure S5 -

  Fig. S5. Normal expression of Ca²⁺ signalling-related proteins in TRIC-B-knockout neonatal lungs. Total homogenates were prepared from the neonatal lungs and analysed by immunoblotting. (A) Representative reactivities using antibodies to P2Y₂, PAR4, IP₃R1, IP₃R3, Gαq/11, PLCβ3, SERCA2 and actin. (B) No differences were detected in expression of the major Ca²⁺ signalling-related proteins between the mutant and wild-type lungs.

• Supplemental Figure S6 -

  Fig. S6. Normal Ca²⁺ responses in alveolar type I cells from TRIC-B-knockout mice. Fura-2 Ca²⁺ imaging in cultured alveolar type I cells. Type I cells showed ATP-evoked Ca²⁺ transients in normal bathing solution (A) but not in Ca²⁺-free solution (B), in a manner fully consistent with the previous report that P2X₄ receptors are expressed in type I cells (Qiao et al., 2003). TRIC-B-knockout type I cells showed normal resting Ca²⁺ levels (C) and normal ATP and ionomycin-induced Ca²⁺ responses (D). The data represent the mean±s.e.m., and the numbers of cells examined are shown in parentheses.

  Reference

  Qiao, R., Zhou, B., Liebler, J. M., Li, X., Crandall, E. D. and Borok, Z. (2003). Identification of three genes of known function expressed by alveolar epithelial type I cells. Am. J. Respir. Cell Mol. Biol. 29, 98-105.

• Supplemental Figure S7 -

  Fig. S7. Normal Ca²⁺ responses in cardiac myocytes from TRIC-B-knockout mice. Fluo-3 Ca²⁺ imaging in cultured cardiac myocytes derived from neonatal mice. Representative traces are shown of imaging data from wild-type (A) and TRIC-B-knockout myocytes (B). Normal peak amplitudes during spontaneous Ca²⁺ oscillations, caffeine-evoked Ca²⁺ transients and SOCE responses were analysed in TRIC-B-knockout myocytes (C). Because TRIC-A channels are predominantly expressed in the neonatal heart (D), the effects of the TRIC-B deficiency are probably minimal in cardiac myocytes. The data represent the mean±s.e.m., and the numbers of cells examined are shown in parentheses.

• Supplemental Figure S8 -

  Fig. S8. Normal Ca²⁺ responses in embryonic fibroblasts from TRIC-B-knockout mice. Fura-2 Ca²⁺ imaging in cultured fibroblasts prepared from E14.5 mice. TRIC-B-knockout fibroblasts showed normal Ca²⁺ transients evoked by ATP and bradykinin (A,B,D) and regular resting Ca²⁺ levels (C). TRIC-B-knockout fibroblasts also showed normal Ca²⁺ responses induced by thapsigargin (TG) and ionomycin (IM), and regular SOCE responses (E,F,G). Because both TRIC channel subtypes are expressed (H), the effects of TRIC-B deficiency are likely to be masked in cultured embryonic fibroblasts. The data represent the mean±s.e.m., and the numbers of cells examined are shown in parentheses.