Figures
- FIGURE 1
Quantitative analysis of differentiation and senescence markers in freshly isolated alveolar type II (ATII) cells from control and idiopathic pulmonary fibrosis (IPF) lungs. Real-time quantification of the expression levels of the following genes in ATII cells harvested from control (n=6) and IPF (n=6) patients: a) aquaporin 5 (AQP5), b) HOP homeobox (HOPX), c) surfactant protein C (SFTPC), d) surfactant protein A1 (SFTPA1), e) zinc finger E-box binding homeobox 1 (ZEB1), f) zinc finger E-box binding homeobox 2 (ZEB2), g) cyclin dependent kinase inhibitor 1A (CDKN1A) and h) cyclin dependent kinase inhibitor 2A (CDKN2A). Data are presented as mean±sd relative expression normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ns: nonsignificant; *: p<0.05; **: p<0.01.
- FIGURE 2
Time-course analysis of differentiation and senescence markers in alveolar type II (ATII) cells upon ex vivo transdifferentiation into alveolar type I (ATI) cells. IPF: idiopathic pulmonary fibrosis. Real-time quantification of the expression levels of the following genes in ATII cells harvested from control (n=6) and IPF (n=6) patients and kept in culture for 2, 4 and 6 days in conditions promoting their transdifferentiation into ATI cells: a) aquaporin 5 (AQP5), b) HOP homeobox (HOPX), c) surfactant protein C (SFTPC), d) surfactant protein A1 (SFTPA1), e) zinc finger E-box binding homeobox 1 (ZEB1), f) zinc finger E-box binding homeobox 2 (ZEB2), g) cyclin dependent kinase inhibitor 1A (CDKN1A) and h) cyclin dependent kinase inhibitor 2A (CDKN2A). Data are presented as mean±sd relative expression normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *: p<0.05; **: p<0.01; ***: p<0.001.
- FIGURE 3
Validation by immunofluorescence of the expression of differentiation and senescence markers in alveolar type II cells upon ex vivo transdifferentiation into alveolar type I cells. SPC: surfactant protein C; IPF: idiopathic pulmonary fibrosis; AQP5: aquaporin 5. a) Quantification of cells expressing pro-SPC at day 2, 4 and 6 of culture in differentiating conditions obtained from control (n=6) and IPF (n=6) patients. b) Representative images of cells expressing pro-SPC (red) after 2 days of culture in differentiating conditions. c) Quantification of cells expressing AQP5 as in (a). d) Representative images of cells expressing AQP5 (red) after 6 days of culture in differentiating conditions. e) Quantification of cells expressing p21 as in (a). f) Representative images of cells expressing p21 as in (d). g) Quantification of cells expressing marker of proliferation Ki-67 as in (a). h) Representative images of cells expressing Ki-67 as in (d). Data are presented as mean±sd percentage of positive cells. *: p<0.05; **: p<0.01; ***: p<0.001. Nuclei are stained in blue with Hoechst 33342. Scale bar: 100 μm.
- FIGURE 4
Effect of miR-200 family members on the differentiation, senescence and proliferation of alveolar type II (ATII) cells from idiopathic pulmonary fibrosis patients. miRNA: microRNA. a) Sequences of the selected miR-200 family members, with indication of the seed sequence and the unique single nucleotide difference between miR-200b/c-3p and miR-200a/141-3p, highlighted in green and red, respectively. b, c) Real-time quantification of the expression of the described targets zinc finger E-box binding homeobox 1 (ZEB1) and 2 (ZEB2) at day 8 after the delivery of the indicated miRNAs. d) Real-time quantification and e) representative images of the expression of aquaporin 5 (AQP5) at day 8 after the delivery of the indicated miRNAs. f) Real-time quantification of the expression of HOP homeobox (HOPX) at day 8 after the delivery of the indicated miRNAs. g) Real-time quantification and h) representative images of the expression of cyclin dependent kinase inhibitor 1A (CDKN1A, p21) at day 8 after the delivery of the indicated miRNAs. i) Real-time quantification of the expression of cyclin dependent kinase inhibitor 2A (CDKN2A) at day 8 after the delivery of the indicated miRNAs. j) Quantification of marker of proliferation Ki-67+ cells at day 8 after the delivery of the indicated miRNAs. Data are presented as mean±sd expressed as fold change over mock (cells transfected with lipid only). ns: nonsignificant; *: p<0.05; **: p<0.01; ***: p<0.001. Nuclei are stained blue with Hoechst 33342. Scale bar: 50 μm.
Supplementary Materials
Supplementary Material
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Supplementary material 00138-2019.SUPPLEMENT
