pro-co2 controller  (Biospherics Inc)

Journal: JCI insight

Article Title: Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.

doi: 10.1172/jci.insight.159331

Figure Lengend Snippet: Figure 1. Hypercapnia limits AT2 cell proliferation in 3D culture organoids. (A) Schematic of experiments designed to coculture AT2 cells isolated from SftpCreERT2 R26REYFP mice (SftpcEYFP AT2) and WT mesenchymal cells. Alveolar organoids were switched to normocapnia (5% CO2; NC) or hypercapnia (20% CO2; HC) media on day 7 and cultured until day 21. (B) Representative images of organoid cultures in normocapnia or hypercapnia. Scale bars: 500 μm. (C) Graph depicts the inhibitory effect of hypercapnia on organoid size. Median with interquartile range. n = 8. (D) Graph depicts the effect of hypercapnia exposure for 21 days on colony forming efficiency (CFE). n = 8. (E) Immunofluorescence analysis of SFTPC (AT2 marker) and Podoplanin (AT1 marker) revealed a reduction in AT2 cell proliferation in organoids exposed to hypercapnia for 14 days relative to normocapnia. Nuclear DNA is stained with DAPI. Scale bars: 50 μm. (C) ANOVA plus Sidak’s multiple comparisons test. (D) Student’s t test. *P < 0.05; ***P < 0.001, ****P < 0.0001.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO2 controller (BioSpherix).

Techniques: Isolation, Cell Culture, Immunofluorescence, Marker, Staining

Journal: JCI insight

Article Title: Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.

doi: 10.1172/jci.insight.159331

Figure Lengend Snippet: Figure 2. Transcriptomic analysis of isolated AT2 cells reveals inhibition of βcat signaling during hypercapnia. (A) Hypercapnia decreases the number of cells expressing Ki67 in the alveolar region of the adult mouse lung exposed to room air (RA) or 10% CO2 (HC) for 21 days, as revealed by immunoflu- orescence. White arrows indicate SPTPC+Ki67+ AT2 cells. Scale bars: 20 μm. (B) Graph depicting the inhibitory effect of hypercapnia exposure for 21 days on proliferation. RA, n = 4; HC, n = 3 mice. Student’s t test. **P < 0.01. (C–F) Bulk RNA-Seq was performed on flow cytometry sorted AT2 cells from mice breathing RA (n = 6) or exposed to HC. Heatmap shows clustering of differentially expressed genes (FDR q < 0.05) in AT2 cells after 7 (n = 5) or 21 (n = 5) days of hypercapnia exposure. (D and E) Volcano plots. (F) GO biological processes. (G–J) Expression of selected DEG (FDR q < 0.05) regulated by hypercap- nia involved in the Wnt/βcat pathway.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO2 controller (BioSpherix).

Techniques: Isolation, Inhibition, Expressing, RNA Sequencing, Flow Cytometry

Journal: JCI insight

Article Title: Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.

doi: 10.1172/jci.insight.159331

Figure Lengend Snippet: Figure 3. Hypercapnia decreases Wnt/βcat signaling in AT2 cells. AT2 cells were isolated from mice exposed to room air (RA) or 10% CO2 (HC) for 21 days. (A) mRNA was isolated, and qPCR was performed. n = 8 mice. (B and C) In situ RNA hybridization showing decreased number of Axin2+ AT2 cells in mice exposed to HC. Yellow arrows indicate Sftpc+Axin2+ AT2 cells. Scale bars: 10 μm. n = 4 mice. (D) Number of lineage-labeled AT2 cells from Axin2CreERT2–TdTom mice determined by flow cytometry. n = 5 mice. Graph shows data from 1 of 3 independent experiments. Student’s t test. *P < 0.05; **P < 0.01.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO2 controller (BioSpherix).

Techniques: Isolation, In Situ, Hybridization, Labeling, Flow Cytometry

Journal: JCI insight

Article Title: Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.

doi: 10.1172/jci.insight.159331

Figure Lengend Snippet: Figure 4. Hypercapnia increases Wnt5a expression in PDGFRα+ fibroblasts. Lung PDGFRα+ fibroblasts were isolated via flow cytometry cell sorting from mice breathing room air (RA) or exposed to 10% CO2 (HC) for 10 days. (A) Expression of Wnt genes in PDGFRα+ fibroblasts as analyzed by population RNA-Seq. n = 3, with cells isolated from 3 mice in each replicate. #FDR q < 0.05). (B–D) mRNA was isolated, and qPCR was performed. (B) Wnt5a (n = 4). (C) Wnt2 (n = 3). (D) MLg2908 mouse lung fibroblast cells were preincubated in the presence or absence of UO126 (10 μM) or PD98059 (10 μM) for 90 minutes and exposed to media equilibrated to NC (5% CO2) or HC (20% CO2) for 24 hours. n = 3. (B and C) Student’s t test. (D) ANOVA plus Sidak’s multiple comparisons test. *P < 0.05; ** P < 0.01.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO2 controller (BioSpherix).

Techniques: Expressing, Isolation, Flow Cytometry, FACS, RNA Sequencing