Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 4. Activation of α7nAChR on AT2 cells promotes alveolar organoid formation. (A) Schematic of organoid coculture of Sftpc lineage–labeled cells (CD45–CD31–EPCAM+tdTomato+) with lung mesenchymal cells (CD45–CD31–EPCAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice and the interven- tion diagram; LPS (1 μg/mL) was added to simulate lung injury in vitro and methyllycaconitine citrate (MLA; α7nAChR antagonist, 10 μmol/L) 15 minutes before GTS-21 (selective α7nAChR agonist, 10 μmol/L) treatment. Times for renewing organoid medium and adding LPS, GTS-21, and MLA are indicated. The culture time varies in different experiments according to purpose. RS, R-Spondin-1. (B) Representative fluorescence images of AT2 organoids captured on day 10. Scale bars: 400 μm. (C) Statistical quantification of the total colony formation efficiency of alveolar organoids. Each individual dot represents 1 experiment in 1 mouse. (D) Statistical quantification of the size of alveolar organoids. Each individual dot represents 1 organoid. (E) Statistical quantifica- tion of total colony formation efficiency of alveolar organoids of different sizes. (F) Representative fluorescence images showing proliferating cells in AT2 organoids derived from the lungs of lineage-tracing mice. Organoids were treated with 5-ethynyl-2′-deoxyuridine (EdU) at an early time point (day 8) for 3 hours in cultures (tdTomato, red; EdU, green; DAPI, blue). Scale bars: 50 μm. One-way ANOVA with Tukey’s post hoc analysis was used in C–E. Data are representative of at least 3 independent experiments and are presented as mean ± SD (*P < 0.05, ***P < 0.001).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Activation Assay, Labeling, Isolation, Knock-Out, In Vitro, Fluorescence, Derivative Assay

Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 6. α7nAChR-driven WNT7B signaling is an essential mediator of α7nAChR-promoted alveolar organoid formation. (A) Schematic of organoid coculture of Sftpc lineage–labeled AT2 cells (CD45–CD31–EPCAM+tdTomato+) isolated from indicated mice, with lung mesenchymal cells (CD45–CD31–EPCAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice, and the intervention diagram. (B) Representative IF images of AT2 organoids. LPS (1 μg/mL) was added to simulate lung injury in vitro, and GTS-21(10 μmol/L) was used to activate α7nAChR in all groups. WNT7B (100 ng/mL) was added as indicated. Scale bars: 400 μm. (C) Statistical quantification of the total colony formation efficiency of alveolar organoids. Each individual dot represents 1 experiment. (D) Statisti- cal quantification of the size of alveolar organoids. Each individual dot represents 1 organoid. One-way ANOVA with Tukey’s post hoc analysis was used in C and D. Data are representative of at least 3 independent experiments and are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Labeling, Isolation, Knock-Out, In Vitro

Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 7. α7nAChR-driven WNT7B signaling is integral for α7nAChR-promoted AT2 cell proliferation and differentiation. (A) Schematic of organoid coculture of Sftpc lineage–labeled cells (CD45–CD31–EPCAM+tdTomato+) isolated from indicated mice, with lung mesenchymal cells (CD45–CD31–EP- CAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice, and the intervention diagram. (B) Effect of selective knockout of α7nAChR on AT2 cells and supplementation of WNT7B (100 ng/mL) on the proliferation of lineage-traced AT2 cells in organoids on day 10, as judged by Ki67 (proliferative marker) (tdTomato, red; Ki67, green; DAPI, blue). Scale bars: 200 μm. (C) Effect of selective knockout of α7nAChR on AT2 cells and supplementation of WNT7B (100 ng/mL) on the differentiation of lineage-traced AT2 cells in organoids on day 12, as judged by PDPN (AT1 marker) (tdTomato, red; PDPN, cyan; DAPI, blue). Scale bars: 200 μm. (D) qPCR analysis of Mki67 from mouse AT2 organoids. (E) qPCR analysis of Pdpn from mouse AT2 organoids. One-way ANOVA with Tukey’s post hoc analysis was used in D and E. Data are presented as mean ± SD (*P < 0.05).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Labeling, Isolation, Knock-Out, Marker

Journal: Drug Design, Development and Therapy

Article Title: MK-4 Ameliorates Diabetic Osteoporosis in Angiogenesis-Dependent Bone Formation by Promoting Mitophagy in Endothelial Cells

doi: 10.2147/DDDT.S503930

Figure Lengend Snippet: MK-4 ameliorates the impairments of type H vessel formation and angiogenesis-dependent bone formation in DOP mice. ( a ) Schematic drawing of the procedures conducted in this study. ( b and c ) Representative flow cytometry plots of CD31 hi Emcn hi ECs (type H ECs). The bar graph showed the quantitation of type H ECs. ( d and e ) Confocal image of CD31 hi and Emcn hi (type H blood vessels) in mice models and histomorphometric quantitation of the area of type H blood vessels. Scale bar: 50μm. ( f and g ) Confocal image of Osterix + (green) osteoprogenitors around CD31 hi vessels (red) and the quantitative analysis. Scale bar: 50μm. ( h ) Typical three-dimensional and two-dimensional coronal images of the tibias were obtained by Micro-CT. ( i ) HE and Masson staining of mice tibias. Scale bar: 200μm or 100μm. ( j ) Statistical analysis of bone histological parameters. ( k and l ) IHC analysis of ALP and RUNX2 in the tibias of mice models. Scale bar: 50μm. Data were presented as mean ± SD (n=5). * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: The cells were subsequently washed and later incubated with PE-conjugated CD45 (E-AB-F1136D, 1:20; Elabscience, Wuhan, China) and APC-conjugated CD31 (E-AB-F1180E, 1:20; Elabscience) antibodies at 4 °C for 45 minutes.

Techniques: Flow Cytometry, Quantitation Assay, Micro-CT, Staining

Journal: Drug Design, Development and Therapy

Article Title: MK-4 Ameliorates Diabetic Osteoporosis in Angiogenesis-Dependent Bone Formation by Promoting Mitophagy in Endothelial Cells

doi: 10.2147/DDDT.S503930

Figure Lengend Snippet: 3-MA partially counteracts the promotion of type H vessel recovery and angiogenesis-dependent bone formation gain by MK-4. ( a and b ) Confocal image of CD31 hi and Emcn hi (termed type H blood vessels) in mice models and histomorphometric quantitation of the area of type H blood vessels. Scale bar: 50μm. ( c and d ) Confocal image of Osterix + (red) osteoprogenitors around Emcn hi vessels (green) and the quantitative analysis. Scale bar: 50μm. ( e ) HE and Masson staining of mice tibias. Scale bar: 200μm or 100μm. ( f ) Statistical analysis of bone histological parameters. ( g and h ) IHC analysis of ALP and RUNX2 in the tibias of mice models. Scale bar: 50μm. Data were presented as mean ± SD (n=5). * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: The cells were subsequently washed and later incubated with PE-conjugated CD45 (E-AB-F1136D, 1:20; Elabscience, Wuhan, China) and APC-conjugated CD31 (E-AB-F1180E, 1:20; Elabscience) antibodies at 4 °C for 45 minutes.

Techniques: Quantitation Assay, Staining