(A) The Uniform Manifold Projection (UMAP) of endothelial cell subtypes from single-cell RNA sequencing (scRNA-seq) of whole mouse lung at homeostasis (n=1). (A’, A'') Atf3 transcript expression in …
(A) Both CAP1_As and CAP1_Bs express general pulmonary capillary EC marker Gpihbp1. (B) The CAP1_B cluster is defined by the expression of transcription factor Atf3. (C) CAP2s express the known …
(A) CAP1s express marker gene Gpihbp1 across both A and B subclusters. (B) CAP1_Bs are additionally defined by the expression of the transcription factor Atf3. (C) CAP2s are defined by the …
(A) The Uniform Manifold Projection (UMAP) dimension reduction plots from the integration of control and influenza scRNA-seq data, with colored dots representing cellular contributions to each …
(A) Schematic of the experimental setup to investigate Atf3-expressing cells during regeneration after H1N1. Atf3CreERT2; ROSA26LSL-tdTomato (Atf3-tdTm) adult mice (5–13 weeks old) received 200 …
(A) Atf3CreERT2; ROSA26LSL-tdTomato (Atf3-tdTm) adult mice treated with tamoxifen 8 days after H1N1 influenza injury show robust tdTomato expression at 22 days post-injury (dpi). (B) Atf3-tdTm adult …
(A) Immunostaining for the AT2 cell marker DC-LAMP (LAMP3), red, and the alveolar epithelial type 1 (AT1) cell marker HOPX, green, in Atf3CreERT2; ROSA26LSL-tdTomato adult mice treated with …
(A) A tile scan of immunofluorescence (IF) for the nuclear endothelial marker ERG, cytoplasmic endothelial marker Endomucin-1, and tdTomato in Atf3-tdTm animals at homeostasis show that …
(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) adult mice (5–15 weeks old) received 200 mg/kg …
(A) Example 20 X image taken from a lung tissue section from a Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mouse at 90 days post influenza (dpi). Immunofluorescence (IF) was performed …
(A) Representative images of Atf3EC-WT and (B) Atf3EC-KO lung tissue at 21 days post influenza stained with hematoxylin and eosin (H&E) demonstrate areas of tissue damage caused by H1N1 infection in …
(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato mice (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mice were given tamoxifen at 200 mg/kg by …
(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato mice (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mice were given 200 mg/kg tamoxifen by oral …
(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato (Atf3EC-WT, n=4) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO, n=3) adult mice received 200 mg/kg tamoxifen …
(A) qRT-PCR for Atf3 demonstrates lower expression in Atf3EC-KO (n=9) compared to Atf3EC-WT (n=8) mouse lung endothelial cells. Genes shown to be downregulated by RNA sequencing also show lower …
(A) Immunofluorescence (IF) for tdTomato (magenta) and DAPI (blue) at 14 days post-H1N1 shows an expected distribution of tdTomato+ ECs throughout the alveolar space in Atf3EC-WT animals. (A’) …
(A) Representative image of lung tissue from an Atf3EC-WT mouse at 21 dpi with immunofluorescence (IF) for (A’) cytoplasmic endothelial marker Endomucin-1 (EndoM1, green), (A’’) tdTomato (magenta), …
(A) Representative image from lung tissue section of an Atf3EC-WT mouse at 21 dpi. Immunofluorescence (IF) analysis was performed for the pan-epithelial marker NKX2-1 (white), the AT2 cell marker …
(A) Representative image from lung tissue section of an Atf3EC-WT mouse at 21 dpi. RNAscope was performed for the CAP2 marker Car4 (white), and IF analysis was performed for tdTomato (red) and the …
(A) Compared to Atf3EC-WT mice (n=4), Atf3EC-KO mice (n=4) have significantly decreased EC proliferation (%CD31+/EdU+ of CD45- cells) between 7–14 dpi. Error bars represent standard deviation (SD). …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus, male and female) | Atf3tm1.1(cre/ERT2)Msra (Atf3CreERT2) | Matt Ramer | MGI: 6360893 | PMID:26610346 |
Strain, strain background (Mus musculus, male and female) | C57BL/6-Tg(Cdh5-cre/ERT2)1Rha (Cdh5CreERT2) | Taconic | 13073, MGI: 3848982 | PMID:19144989 |
Strain, strain background (Mus musculus, male and female) | Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (ROSA26LSL-tdTomato) | Jackson Laboratory | 007914, MGI: 3809524 | PMID:20023653 |
Strain, strain background (Mus musculus, male and female) | Atf3tm1.1Hai (Atf3lox) | Tsonwin Hai | MGI: 5547956 | PMID:23921126 |
Antibody | tdTomato (goat polyclonal) | Origene | AB8181-200 | 1:50 |
Antibody | RFP (DsRed) (rabbit polyclonal) | Rockland | 600-401-379, RRID:AB_2209751 | 1:50 |
Antibody | ERG (mouse monoclonal) | Abcam | ab214341 | 1:100 |
Antibody | ERG (rabbit monoclonal) | Abcam | ab92513 RRID:AB_2630401 | 1:100 |
Antibody | Endomucin-1 (goat polyclonal) | R&D Systems | AF4666 RRID:AB_2100035 | 1:500 |
Antibody | LAMP3 (rat monoclonal) | Novus Biologicals | DDX0191P-100 RRID:AB_2827532 | 1:100 |
Antibody | HOPX (mouse monoclonal) | Santa Cruz Biotechnology | sc-398703 RRID:AB_2687966 | 1:100 |
Antibody | NKX2-1 (TTF1) (rabbit monoclonal) | Abcam | ab76013 RRID:AB_1310784 | 1:50 |
Antibody | PDPN (mouse monoclonal) | Abcam | ab10288 RRID:AB_297027 | 1:100 |
Antibody | CD45-PerCP-Cy5.5 (rat monoclonal) | Thermo Fisher | 45-0451-82 RRID:AB_1107002 | 1:200 |
Antibody | CD31-PE-Cy7 (rat monoclonal) | Thermo Fisher | 25-0311-82 RRID:AB_2716949 | 1:100 |
Chemical compound, drug | PR8-GP33 H1N1 influenza | Dr. E. John Wherry | PMID:23516357 | |
Chemical compound, drug | Tamoxifen | Sigma-Aldrich | T5648 | |
Commercial assay or kit | Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit | Thermo Fisher | C10632 | |
Commercial assay or kit | RNAscope Fluorescent Multiplex Reagent Kit v2 | ACD | 323100 | |
Commercial assay or kit | PureLink RNA Micro Kit | Thermo Fisher | 12183–016 | |
Commercial assay or kit | Bioanalyzer High Sensitivity RNA Pico Kit | Agilent | 5067–1513 | |
Commercial assay or kit | NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina | New England Biolabs | E6420 | |
Commercial assay or kit | Bioanalyzer High Sensitivity DNA Kit | Agilent | 5067–4626 | |
Other | RNAscope probe mm-Car4-C3 | ACD | 468421-C3 | Probe to detect Car4 RNA; see Figure 5—figure supplement 3 and Materials and methods: RNAscope analysis |
Other | RNAscope probe mm-Pdgfra-C2 | ACD | 480661-C2 | Probe to detect Pdgfra RNA; see Figure 2—figure supplement 2 and Materials and methods: RNAscope analysis |