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rabbit anti pex5 polyclonal antibody  (Novus Biologicals)


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    Novus Biologicals rabbit anti pex5 polyclonal antibody
    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of <t>Pex5</t> in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .
    Rabbit Anti Pex5 Polyclonal Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nbp1/PEX5+Antibody/pmc13657169-2-0-5
    Average 91 stars, based on 6 article reviews
    rabbit anti pex5 polyclonal antibody - by Bioz Stars, 2026-10
    91/100 stars

    Images

    1) Product Images from "Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells"

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    Journal: iScience

    doi: 10.1016/j.isci.2026.117658

    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .
    Figure Legend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Techniques Used: Blocking Assay, Staining, Analysis


    Figure Legend Snippet:

    Techniques Used: Cell-Signaling, Magnetic Beads, Virus, Recombinant, ROS Assay, Reverse Transcription, SYBR Green Assay, Staining, Plasmid Preparation, Flow Cytometry, Western Blot, Expressing, Control, Software

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    Chromatin Immunoprecipitation:

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    Article Snippet: .. ChIP assay was performed using the ChromataChIP Kit (Novus Biologicals) (cat: NBP1-71709) following the manufacturer's protocol. ..

    Cell-Signaling:

    Article Title: Cell volume regulation in metazoans involves phosphorylation of TSC22D proteins.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-TSC22D1 Proteintech Cat. No.: 10214-1-Ig; RRID: AB_2272193 Anti-TSC22D2 Novus Biologicals Cat. No.: NBP1-81491; RRID: AB_11035526 Anti-TSC22D4 Abnova Cat. No.: H00081628-M07; RRID: AB_733263 Anti-phospho-WNK1 (Thr60) R&D Systems Cat. No.: MAB4720; RRID: AB_3658354 Anti-WNK1 abcam Cat. No.: 137687; RRID: AB_11001564 Anti-NRBP1 Abnova Cat. No.: H00029959-M02: RRID: AB_606683 Anti-phospho-SPAK (Ser373)/phospho-OSR1 (Ser325) Millipore Cat. No.: 07-2273; RRID: AB_11213418 Anti-SPAK Cell Signaling Technology Cat. No.: 2281; RRID: AB_2196951 Anti-OXSR1 Sigma-Aldrich Cat. No.: HPA008237; RRID: AB_1079540 Anti-GAPDH (14C10) Cell Signaling Technology Cat. No.: 2118; RRID: AB_561053 Anti-β-Actin (8H10D10) Cell Signaling Technology Cat. No.: 3700; RRID: AB_2242334 Anti-phospho-SAPK/JNK (Thr183/Tyr185) Cell Signaling Technology Cat. No.: 9251; RRID: AB_331659 Anti-FLAG M2 Sigma-Aldrich Cat. No.: F1804; RRID: AB_262044 Anti-HA-tag (6E2) Cell Signaling Technology Cat. No.: 2367; RRID: AB_10691311 Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, HRP Invitrogen Cat. No.: A16072; RRID: AB_2534745 Goat Anti-Rabbit (H + L)-HRP Conjugate Bio-Rad Cat. No.: 170-6515; RRID: AB_11125142 Bacterial and virus strains pLJM1-based 3rd gen lentiviral vector Addgene ID: 19319 One ShotTM Stbl3TM Chemically Competent E. coli Thermo Fisher Scientific Cat. No. C737303 Biological samples .. Chemicals, peptides, and recombinant proteins WNK-IN-11 Selleckchem Cat. No. S0015 WNK463 Selleckchem Cat. No. S8358 Nystatin Thermo Fisher Scientific Cat. No. J62486.06 JNK-IN-7 Cayman Chemical Cat. No. 28831 Anti-FLAG M2 magnetic beads Sigma-Aldrich Cat. No. M8823 Pierce Protein A/G Plus agarose beads Thermo Fisher Scientific Cat. No. 20423 PEG 20,000 Da Thermo Fisher Scientific Cat. No. A17925.30 Ficoll 400,000 Da Sigma-Aldrich Cat. No. F4375 Dextran 40,000 Da Sigma-Aldrich Cat. No. 31389 desthiobiotin-ATP Thermo Fisher Scientific Cat. No. 88311 lambda protein phosphatase New England Biolabs Cat. No. P0753S CellMask Deep Red Thermo Fisher Scientific Cat. No. C10046 Hoechst 33342 solution Thermo Fisher Scientific Cat. No. 62249 Critical commercial assays X-tremeGENE 9 DNA transfection reagent Roche Cat. No. 6365779001 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat. No. 23227 (Continued on next page) Cell Reports 45, 118051, October 27, 2026 23

    Virus:

    Article Title: Cell volume regulation in metazoans involves phosphorylation of TSC22D proteins.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-TSC22D1 Proteintech Cat. No.: 10214-1-Ig; RRID: AB_2272193 Anti-TSC22D2 Novus Biologicals Cat. No.: NBP1-81491; RRID: AB_11035526 Anti-TSC22D4 Abnova Cat. No.: H00081628-M07; RRID: AB_733263 Anti-phospho-WNK1 (Thr60) R&D Systems Cat. No.: MAB4720; RRID: AB_3658354 Anti-WNK1 abcam Cat. No.: 137687; RRID: AB_11001564 Anti-NRBP1 Abnova Cat. No.: H00029959-M02: RRID: AB_606683 Anti-phospho-SPAK (Ser373)/phospho-OSR1 (Ser325) Millipore Cat. No.: 07-2273; RRID: AB_11213418 Anti-SPAK Cell Signaling Technology Cat. No.: 2281; RRID: AB_2196951 Anti-OXSR1 Sigma-Aldrich Cat. No.: HPA008237; RRID: AB_1079540 Anti-GAPDH (14C10) Cell Signaling Technology Cat. No.: 2118; RRID: AB_561053 Anti-β-Actin (8H10D10) Cell Signaling Technology Cat. No.: 3700; RRID: AB_2242334 Anti-phospho-SAPK/JNK (Thr183/Tyr185) Cell Signaling Technology Cat. No.: 9251; RRID: AB_331659 Anti-FLAG M2 Sigma-Aldrich Cat. No.: F1804; RRID: AB_262044 Anti-HA-tag (6E2) Cell Signaling Technology Cat. No.: 2367; RRID: AB_10691311 Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, HRP Invitrogen Cat. No.: A16072; RRID: AB_2534745 Goat Anti-Rabbit (H + L)-HRP Conjugate Bio-Rad Cat. No.: 170-6515; RRID: AB_11125142 Bacterial and virus strains pLJM1-based 3rd gen lentiviral vector Addgene ID: 19319 One ShotTM Stbl3TM Chemically Competent E. coli Thermo Fisher Scientific Cat. No. C737303 Biological samples .. Chemicals, peptides, and recombinant proteins WNK-IN-11 Selleckchem Cat. No. S0015 WNK463 Selleckchem Cat. No. S8358 Nystatin Thermo Fisher Scientific Cat. No. J62486.06 JNK-IN-7 Cayman Chemical Cat. No. 28831 Anti-FLAG M2 magnetic beads Sigma-Aldrich Cat. No. M8823 Pierce Protein A/G Plus agarose beads Thermo Fisher Scientific Cat. No. 20423 PEG 20,000 Da Thermo Fisher Scientific Cat. No. A17925.30 Ficoll 400,000 Da Sigma-Aldrich Cat. No. F4375 Dextran 40,000 Da Sigma-Aldrich Cat. No. 31389 desthiobiotin-ATP Thermo Fisher Scientific Cat. No. 88311 lambda protein phosphatase New England Biolabs Cat. No. P0753S CellMask Deep Red Thermo Fisher Scientific Cat. No. C10046 Hoechst 33342 solution Thermo Fisher Scientific Cat. No. 62249 Critical commercial assays X-tremeGENE 9 DNA transfection reagent Roche Cat. No. 6365779001 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat. No. 23227 (Continued on next page) Cell Reports 45, 118051, October 27, 2026 23

    Plasmid Preparation:

    Article Title: Cell volume regulation in metazoans involves phosphorylation of TSC22D proteins.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-TSC22D1 Proteintech Cat. No.: 10214-1-Ig; RRID: AB_2272193 Anti-TSC22D2 Novus Biologicals Cat. No.: NBP1-81491; RRID: AB_11035526 Anti-TSC22D4 Abnova Cat. No.: H00081628-M07; RRID: AB_733263 Anti-phospho-WNK1 (Thr60) R&D Systems Cat. No.: MAB4720; RRID: AB_3658354 Anti-WNK1 abcam Cat. No.: 137687; RRID: AB_11001564 Anti-NRBP1 Abnova Cat. No.: H00029959-M02: RRID: AB_606683 Anti-phospho-SPAK (Ser373)/phospho-OSR1 (Ser325) Millipore Cat. No.: 07-2273; RRID: AB_11213418 Anti-SPAK Cell Signaling Technology Cat. No.: 2281; RRID: AB_2196951 Anti-OXSR1 Sigma-Aldrich Cat. No.: HPA008237; RRID: AB_1079540 Anti-GAPDH (14C10) Cell Signaling Technology Cat. No.: 2118; RRID: AB_561053 Anti-β-Actin (8H10D10) Cell Signaling Technology Cat. No.: 3700; RRID: AB_2242334 Anti-phospho-SAPK/JNK (Thr183/Tyr185) Cell Signaling Technology Cat. No.: 9251; RRID: AB_331659 Anti-FLAG M2 Sigma-Aldrich Cat. No.: F1804; RRID: AB_262044 Anti-HA-tag (6E2) Cell Signaling Technology Cat. No.: 2367; RRID: AB_10691311 Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, HRP Invitrogen Cat. No.: A16072; RRID: AB_2534745 Goat Anti-Rabbit (H + L)-HRP Conjugate Bio-Rad Cat. No.: 170-6515; RRID: AB_11125142 Bacterial and virus strains pLJM1-based 3rd gen lentiviral vector Addgene ID: 19319 One ShotTM Stbl3TM Chemically Competent E. coli Thermo Fisher Scientific Cat. No. C737303 Biological samples .. Chemicals, peptides, and recombinant proteins WNK-IN-11 Selleckchem Cat. No. S0015 WNK463 Selleckchem Cat. No. S8358 Nystatin Thermo Fisher Scientific Cat. No. J62486.06 JNK-IN-7 Cayman Chemical Cat. No. 28831 Anti-FLAG M2 magnetic beads Sigma-Aldrich Cat. No. M8823 Pierce Protein A/G Plus agarose beads Thermo Fisher Scientific Cat. No. 20423 PEG 20,000 Da Thermo Fisher Scientific Cat. No. A17925.30 Ficoll 400,000 Da Sigma-Aldrich Cat. No. F4375 Dextran 40,000 Da Sigma-Aldrich Cat. No. 31389 desthiobiotin-ATP Thermo Fisher Scientific Cat. No. 88311 lambda protein phosphatase New England Biolabs Cat. No. P0753S CellMask Deep Red Thermo Fisher Scientific Cat. No. C10046 Hoechst 33342 solution Thermo Fisher Scientific Cat. No. 62249 Critical commercial assays X-tremeGENE 9 DNA transfection reagent Roche Cat. No. 6365779001 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat. No. 23227 (Continued on next page) Cell Reports 45, 118051, October 27, 2026 23



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    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Journal: iScience

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    doi: 10.1016/j.isci.2026.117658

    Figure Lengend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Article Snippet: Rabbit anti-PEX5 polyclonal antibody , Novus , NBP1-87185; RRID: AB_11023925.

    Techniques: Blocking Assay, Staining, Analysis

    Journal: iScience

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    doi: 10.1016/j.isci.2026.117658

    Figure Lengend Snippet:

    Article Snippet: Rabbit anti-PEX5 polyclonal antibody , Novus , NBP1-87185; RRID: AB_11023925.

    Techniques: Cell-Signaling, Magnetic Beads, Virus, Recombinant, ROS Assay, Reverse Transcription, SYBR Green Assay, Staining, Plasmid Preparation, Flow Cytometry, Western Blot, Expressing, Control, Software

    Embryonic day 12.5 (E12.5) Svep1 +/+ + lung explant culture time-lapse images over 48 hr showing normal branching morphogenesis.

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    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: Embryonic day 12.5 (E12.5) Svep1 +/+ + lung explant culture time-lapse images over 48 hr showing normal branching morphogenesis.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques:

    Time-lapse images of embryonic day 12.5 (E12.5) Svep1 -/- lung explant culture over 48 hr demonstrating aberrant branching at the tips and edges of the lung, primarily in the form of trifurcations.

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: Time-lapse images of embryonic day 12.5 (E12.5) Svep1 -/- lung explant culture over 48 hr demonstrating aberrant branching at the tips and edges of the lung, primarily in the form of trifurcations.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques:

    Time-lapse images of embryonic day 12.5 (E12.5) Svep1 -/- lung explant culture over 48 hr show ectopic branching on the left lobe.

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: Time-lapse images of embryonic day 12.5 (E12.5) Svep1 -/- lung explant culture over 48 hr show ectopic branching on the left lobe.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques:

    Control embryonic day 12.5 (E12.5) Svep1 +/+ + time-lapse images for the rSVEP1-treated lungs ( <xref ref-type=Video 5 ), showing normal branching." width="100%" height="100%">

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: Control embryonic day 12.5 (E12.5) Svep1 +/+ + time-lapse images for the rSVEP1-treated lungs ( Video 5 ), showing normal branching.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques:

    Time-lapse images of embryonic day 12.5 (E12.5) Svep1 +/+ + lung explants treated with rSVEP1 protein for 48 hr, demonstrating decreased secondary branching.

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: Time-lapse images of embryonic day 12.5 (E12.5) Svep1 +/+ + lung explants treated with rSVEP1 protein for 48 hr, demonstrating decreased secondary branching.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques:

    ( a ) In situ hybridization of lungs from embryonic day 14.5 (E14.5) wild-type embryos showed strong Svep1 mRNA expression in the mesenchyme at the lung periphery and around bifurcating distal airway tips (right inset outlined in red, arrowhead). ( b ) Arrowhead shows mesenchymal expression of SVEP1 protein in normal E12.5 lung. White arrows indicate the location of the SVEP1 protein on the epithelial membrane stained with ECAD (purple) in a distal airway bud. ( c ) SVEP1 expression is strong in the mesenchyme toward the lobe tip of the E14.5 lungs and branching cleft sites (arrowheads). ( d ) SVEP1 protein localizes in the lung parenchyma, including the primary septa (upper box) and adjacent to the proximal airway epithelium (lower box). ( e ) microCT scan of E18.5 embryos showing multiple defects, including thinning of the diaphragm and lung hypoplasia (red arrows). ( f ) Ventral view of E18.5 of Svep1 +/+ and Svep1 ‐/- lungs. The arrowheads indicate the tips of the lung lobes, which are short and rounded (highlight in black) in Svep1 -/- mice and have a cauliflower morphology. ( g ) Hematoxylin and eosin (H&E)-stained lung section embryos demonstrate that E16.5 lungs from Svep1 -/- embryos are smaller and have irregular lobe edges. Black arrowheads indicate left lobes with highlighted surface. ( h ) The red arrowheads show the dorsal side of the E18 lungs, whereas the black arrows highlight the ventral side. Svep1 -/- embryos show defective saccular development mainly at the edge on the ventral side of the lung (inserts arrowheads). Co-localization of ECAD and SOX9 confirms high expression of SOX9 in distal airways (white arrowheads) in the lung of Svep1 -/- embryos at E18.5. ( i ) Plots showing Sox9 relative mRNA expression at E14.5, E16.5, and E18.5. mRNA expression is significantly higher at E18.5 in Svep1 -/- embryos (mean ± SEM; Wilcoxon rank-sum test; p<0.05). Right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 50 μm ( a–d ), 100 μm. Figure 1—source data 1. qPCR data for Sox9 expression at E14.5, E16.5, and E18.5 .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) In situ hybridization of lungs from embryonic day 14.5 (E14.5) wild-type embryos showed strong Svep1 mRNA expression in the mesenchyme at the lung periphery and around bifurcating distal airway tips (right inset outlined in red, arrowhead). ( b ) Arrowhead shows mesenchymal expression of SVEP1 protein in normal E12.5 lung. White arrows indicate the location of the SVEP1 protein on the epithelial membrane stained with ECAD (purple) in a distal airway bud. ( c ) SVEP1 expression is strong in the mesenchyme toward the lobe tip of the E14.5 lungs and branching cleft sites (arrowheads). ( d ) SVEP1 protein localizes in the lung parenchyma, including the primary septa (upper box) and adjacent to the proximal airway epithelium (lower box). ( e ) microCT scan of E18.5 embryos showing multiple defects, including thinning of the diaphragm and lung hypoplasia (red arrows). ( f ) Ventral view of E18.5 of Svep1 +/+ and Svep1 ‐/- lungs. The arrowheads indicate the tips of the lung lobes, which are short and rounded (highlight in black) in Svep1 -/- mice and have a cauliflower morphology. ( g ) Hematoxylin and eosin (H&E)-stained lung section embryos demonstrate that E16.5 lungs from Svep1 -/- embryos are smaller and have irregular lobe edges. Black arrowheads indicate left lobes with highlighted surface. ( h ) The red arrowheads show the dorsal side of the E18 lungs, whereas the black arrows highlight the ventral side. Svep1 -/- embryos show defective saccular development mainly at the edge on the ventral side of the lung (inserts arrowheads). Co-localization of ECAD and SOX9 confirms high expression of SOX9 in distal airways (white arrowheads) in the lung of Svep1 -/- embryos at E18.5. ( i ) Plots showing Sox9 relative mRNA expression at E14.5, E16.5, and E18.5. mRNA expression is significantly higher at E18.5 in Svep1 -/- embryos (mean ± SEM; Wilcoxon rank-sum test; p<0.05). Right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 50 μm ( a–d ), 100 μm. Figure 1—source data 1. qPCR data for Sox9 expression at E14.5, E16.5, and E18.5 .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: In Situ Hybridization, Expressing, Membrane, Staining, Morphology

    ( a–b ) Svep1 in situ hybridization in embryonic day 11.5 (E11.5) and E12.5 embryos. Arrowheads indicate high expression of Svep1 at the distal tip of right medial (RMe) and accessory (RAc) lobes (outlined in red) and the left lobe. Arrows show Svep1 expression along and between early airway buds (outlined in red). ( b ) Arrows mark the expression of SVEP1 (green) adjacent to epithelial cells (ECAD; purple) in E14.5 normal lung. ( c ) In situ hybridization showing Svep1 expression in wild-type E16.5 lungs. The area outlined in the left panel is shown at higher magnification in the right-hand panel to highlight airways. Svep1 expression is stronger at the edges of the lung (arrowheads). SVEP1 protein localized next to distal airways (arrowheads) and microvasculature (arrows). ( d ) Svep1 protein or mRNA is seen in the mesenchyme at E18.5 and is highlighted in the primary septum. ( e ) RT-qPCR for Svep1 expression showing relative fold change at E14.5, E16.5, and E18.5. ( f ) Normal and Svep1 -/- embryos at E12.5, E15.5, and E18.5 show edema and cranial, limb, and tail developmental defects increasing in mutants over time. ( g ) Svep1 mRNA is absent in E18.5 mutants. ( h ) SOX9 and KI67 mark proliferating epithelial progenitor cells in E18.5 lungs, mainly at the lung periphery in Svep1 -/- mutants. Scale bars: 100 µm ( a , c , d , h ); 200 µm ( b ). Figure 1—figure supplement 1—source data 1. qPCR expression data for Svep1 at E14.5, E16.5, E18.5 .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a–b ) Svep1 in situ hybridization in embryonic day 11.5 (E11.5) and E12.5 embryos. Arrowheads indicate high expression of Svep1 at the distal tip of right medial (RMe) and accessory (RAc) lobes (outlined in red) and the left lobe. Arrows show Svep1 expression along and between early airway buds (outlined in red). ( b ) Arrows mark the expression of SVEP1 (green) adjacent to epithelial cells (ECAD; purple) in E14.5 normal lung. ( c ) In situ hybridization showing Svep1 expression in wild-type E16.5 lungs. The area outlined in the left panel is shown at higher magnification in the right-hand panel to highlight airways. Svep1 expression is stronger at the edges of the lung (arrowheads). SVEP1 protein localized next to distal airways (arrowheads) and microvasculature (arrows). ( d ) Svep1 protein or mRNA is seen in the mesenchyme at E18.5 and is highlighted in the primary septum. ( e ) RT-qPCR for Svep1 expression showing relative fold change at E14.5, E16.5, and E18.5. ( f ) Normal and Svep1 -/- embryos at E12.5, E15.5, and E18.5 show edema and cranial, limb, and tail developmental defects increasing in mutants over time. ( g ) Svep1 mRNA is absent in E18.5 mutants. ( h ) SOX9 and KI67 mark proliferating epithelial progenitor cells in E18.5 lungs, mainly at the lung periphery in Svep1 -/- mutants. Scale bars: 100 µm ( a , c , d , h ); 200 µm ( b ). Figure 1—figure supplement 1—source data 1. qPCR expression data for Svep1 at E14.5, E16.5, E18.5 .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: In Situ Hybridization, Expressing, Quantitative RT-PCR

    ( a ) UMAP clustering showing matrix fibroblast cells have the highest expression of Svep1 (reddish orange). ( b ) Clusters deficient in mutant cells compared to cells from wild-type samples are shaded in yellow. Clusters with enrichment of cells from mutant lung samples are shaded in blue.

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) UMAP clustering showing matrix fibroblast cells have the highest expression of Svep1 (reddish orange). ( b ) Clusters deficient in mutant cells compared to cells from wild-type samples are shaded in yellow. Clusters with enrichment of cells from mutant lung samples are shaded in blue.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Expressing, Mutagenesis

    ( a ) Co-localization of epithelial (ECAD) and epithelial progenitor (SOX9) markers showing distal airways in lungs of embryonic day 16.5 (E16.5) mice. ( a' ) The blue highlights in inserts represent the distal tip of the normal and mutant RAc lung lobes, with the mutant left lobe showing multiple branching. ( a'' ) Inserts illustrate airway density at the lung periphery in wild-type (WT) and mutants, with mutants having higher density. ( a''' ) In Svep1 -/- lungs, distal airway tips are organized in packed airway rosettes on the ventral side of the lung lobe. ( b ) Illustration of the three branching modes that pattern the airways of the lung (domain branching, planar bifurcation, orthogonal bifurcation) and distal trifurcation. This panel was created using BioRender.com . ( c–f ) Representative Z-stack images show SOX9 immunofluorescence. ( c ) E13.5 whole lungs of normal and Svep1 -/- embryos. The images show Svep1 -/- lungs with abnormal RCa lobe morphology (arrowheads), RAc lobe distal tip branching (arrows), and secondary branching patterning (orange arrowheads). ( d ) Pictures of E13.5 Svep1 +/+ and Svep1 -/- embryos showing ectopic branching/budding anomalies in mutant embryos (areas outlined in pink in lower inserts). ( e ) E14.5 left lung lobes of Svep1 +/+ and Svep1 ‐/‐ embryos. SOX9 + buds highlighted in pink and cartoon show airway tip arrangement in normal and mutants. Arrow shows defective tip arrangement resulting in abnormal lung tip morphology in Svep1 -/- embryos. ( f ) Cleared tissue immunofluorescence of SOX9 in the left lung lobe at E14.5 reveals airway disorganization in Svep1 -/- embryos vs Svep1 +/+ embryos. Distal lung tips highlighted show branching trifurcations in the Svep1 -/- embryo (white arrow) and aberrant distal tip airway. ( g ) Representation of the left lung lobe Z-stack for distal airway quantification. Whole lungs of Svep1 -/- embryos at E14.5 have a statistically significant increase in the total number of SOX9 positive airway tips and ratio of tip and area compared to wild-type embryos (mean ± SEM; two-tailed unpaired t-test; p<0.05). This panel was created using BioRender.com . Right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 100 μm ( a–e ). Figure 2—source data 1. Counts of Sox9 positive airway tips in lungs from wild type and mutant Svep1 embryos at E14.5 .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Co-localization of epithelial (ECAD) and epithelial progenitor (SOX9) markers showing distal airways in lungs of embryonic day 16.5 (E16.5) mice. ( a' ) The blue highlights in inserts represent the distal tip of the normal and mutant RAc lung lobes, with the mutant left lobe showing multiple branching. ( a'' ) Inserts illustrate airway density at the lung periphery in wild-type (WT) and mutants, with mutants having higher density. ( a''' ) In Svep1 -/- lungs, distal airway tips are organized in packed airway rosettes on the ventral side of the lung lobe. ( b ) Illustration of the three branching modes that pattern the airways of the lung (domain branching, planar bifurcation, orthogonal bifurcation) and distal trifurcation. This panel was created using BioRender.com . ( c–f ) Representative Z-stack images show SOX9 immunofluorescence. ( c ) E13.5 whole lungs of normal and Svep1 -/- embryos. The images show Svep1 -/- lungs with abnormal RCa lobe morphology (arrowheads), RAc lobe distal tip branching (arrows), and secondary branching patterning (orange arrowheads). ( d ) Pictures of E13.5 Svep1 +/+ and Svep1 -/- embryos showing ectopic branching/budding anomalies in mutant embryos (areas outlined in pink in lower inserts). ( e ) E14.5 left lung lobes of Svep1 +/+ and Svep1 ‐/‐ embryos. SOX9 + buds highlighted in pink and cartoon show airway tip arrangement in normal and mutants. Arrow shows defective tip arrangement resulting in abnormal lung tip morphology in Svep1 -/- embryos. ( f ) Cleared tissue immunofluorescence of SOX9 in the left lung lobe at E14.5 reveals airway disorganization in Svep1 -/- embryos vs Svep1 +/+ embryos. Distal lung tips highlighted show branching trifurcations in the Svep1 -/- embryo (white arrow) and aberrant distal tip airway. ( g ) Representation of the left lung lobe Z-stack for distal airway quantification. Whole lungs of Svep1 -/- embryos at E14.5 have a statistically significant increase in the total number of SOX9 positive airway tips and ratio of tip and area compared to wild-type embryos (mean ± SEM; two-tailed unpaired t-test; p<0.05). This panel was created using BioRender.com . Right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 100 μm ( a–e ). Figure 2—source data 1. Counts of Sox9 positive airway tips in lungs from wild type and mutant Svep1 embryos at E14.5 .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Mutagenesis, Immunofluorescence, Morphology, Tissue, Two Tailed Test

    ( a ) SOX9 immunostaining of embryonic day 13.5 (E13.5) lung lobe edges highlights (pink) Svep1 branching and bifurcation anomalies at the lung periphery. The blue highlights denote branching domain buds. ( b ) Immunofluorescence images of E14.5 and E15.5 lung lobes showing distal airways in both wild-type and Svep1 -/- embryos. Arrows indicate defective branching in Svep1 -/- embryos. ( c ) Graph of the ratio of length to width for individual E14.5 lobes. Svep1 +/+ in pink; Svep1 -/- in blue. ( d ) Graph of the ratio of length to width for individual E15.5 lobes. Svep1 +/+ in pink; Svep1 -/- in blue. ( e ) DAPI staining of E15.5 lung lobes displays the gross morphology of the lung surface for both Svep1 +/+ and Svep1 -/- specimens. Average lobe length-to-width ratio ± SEM; p<0.01; n≥5. Lobe abbreviations: right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 100 µm. Figure 2—figure supplement 1—source data 1. Ratio of length to width in lungs Svep1 wild type and mutant embryos at E14.5 . Figure 2—figure supplement 1—source data 2. Ratio of length to width in lungs Svep1 wild type and mutant embryos at E15.5 .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) SOX9 immunostaining of embryonic day 13.5 (E13.5) lung lobe edges highlights (pink) Svep1 branching and bifurcation anomalies at the lung periphery. The blue highlights denote branching domain buds. ( b ) Immunofluorescence images of E14.5 and E15.5 lung lobes showing distal airways in both wild-type and Svep1 -/- embryos. Arrows indicate defective branching in Svep1 -/- embryos. ( c ) Graph of the ratio of length to width for individual E14.5 lobes. Svep1 +/+ in pink; Svep1 -/- in blue. ( d ) Graph of the ratio of length to width for individual E15.5 lobes. Svep1 +/+ in pink; Svep1 -/- in blue. ( e ) DAPI staining of E15.5 lung lobes displays the gross morphology of the lung surface for both Svep1 +/+ and Svep1 -/- specimens. Average lobe length-to-width ratio ± SEM; p<0.01; n≥5. Lobe abbreviations: right cranial (RCr), right caudal (RCa), right medial (RMe), and right accessory (RAc) lobes. Scale bars: 100 µm. Figure 2—figure supplement 1—source data 1. Ratio of length to width in lungs Svep1 wild type and mutant embryos at E14.5 . Figure 2—figure supplement 1—source data 2. Ratio of length to width in lungs Svep1 wild type and mutant embryos at E15.5 .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Immunostaining, Immunofluorescence, Staining, Morphology, Mutagenesis

    ( a ) Compilation of a Z-stack image series for tissue-cleared whole RAc lung lobes labeled with ECAD (green) and SOX9 (purple) highlights disorganized branching in Svep1 -/- embryos at E14.5. Arrows (lower right) point to branching airways with rosette-like arrangements and ectopic airway tips growing in random directions. Arrowheads (left panels) at the right caudal (RCa) lobe tips indicate a single tip in the lobe of Svep1 +/+ embryos but multi-branched tips in Svep1 knockout embryos. ( b ) Schematic illustrating representative domain (blue), planar (green), and orthogonal (red) branching program types of Svep1 +/+ and Svep1 -/- RAc lobes. Lungs of Svep1 -/- embryos lack planar bifurcations and have many trifurcating branch tips (red highlighted in black). ( c–d ) Graphs displaying the percentages of branching modes, orthogonal bifurcations, and trifurcations ( c ) and ectopic budding ( d ) in E14.5 RAc lobes of wild-type and Svep1 -/- embryos. ( e ) Representative single Z-stack images of RAc lobes at E14.5, stained with ECAD (green) and SOX9 (purple), show bifurcations (white dotted lines and white arrowhead) in Svep1 +/+ embryos, but trifurcations (white dotted lines and white arrows) in Svep1 -/- mice. ( f ) Single Z-stack image of RAc lobes stained with ECAD (green) and SOX9 (red) displays ectopic domain branching in Svep1 -/- mice (arrow). ( g ) Lung explants from E12.5 embryos after 48 hr in organ culture. Explants from Svep1 +/+ embryos show normal bifurcation (black arrowheads), whereas explants from Svep1 -/- embryos show trifurcations (black arrows) and ectopic budding (red arrowheads) (data are presented as mean ± SEM; two-tailed unpaired t-test; n=4 per genotype) ( c , d ). Scale bars: 100 μm ( a , g ); 50 μm ( e ). Figure 3—source data 1. Percentages of branching modes, orthogonal bifurcations, and trifurcations in right accessor lung lobs of Svep1 wild type and mutant embryo at E14.5 . Figure 3—source data 2. Percentages of ectopic budding in right accessory lung lobes of Svep1 wild type and mutant embryos at E14.5 .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Compilation of a Z-stack image series for tissue-cleared whole RAc lung lobes labeled with ECAD (green) and SOX9 (purple) highlights disorganized branching in Svep1 -/- embryos at E14.5. Arrows (lower right) point to branching airways with rosette-like arrangements and ectopic airway tips growing in random directions. Arrowheads (left panels) at the right caudal (RCa) lobe tips indicate a single tip in the lobe of Svep1 +/+ embryos but multi-branched tips in Svep1 knockout embryos. ( b ) Schematic illustrating representative domain (blue), planar (green), and orthogonal (red) branching program types of Svep1 +/+ and Svep1 -/- RAc lobes. Lungs of Svep1 -/- embryos lack planar bifurcations and have many trifurcating branch tips (red highlighted in black). ( c–d ) Graphs displaying the percentages of branching modes, orthogonal bifurcations, and trifurcations ( c ) and ectopic budding ( d ) in E14.5 RAc lobes of wild-type and Svep1 -/- embryos. ( e ) Representative single Z-stack images of RAc lobes at E14.5, stained with ECAD (green) and SOX9 (purple), show bifurcations (white dotted lines and white arrowhead) in Svep1 +/+ embryos, but trifurcations (white dotted lines and white arrows) in Svep1 -/- mice. ( f ) Single Z-stack image of RAc lobes stained with ECAD (green) and SOX9 (red) displays ectopic domain branching in Svep1 -/- mice (arrow). ( g ) Lung explants from E12.5 embryos after 48 hr in organ culture. Explants from Svep1 +/+ embryos show normal bifurcation (black arrowheads), whereas explants from Svep1 -/- embryos show trifurcations (black arrows) and ectopic budding (red arrowheads) (data are presented as mean ± SEM; two-tailed unpaired t-test; n=4 per genotype) ( c , d ). Scale bars: 100 μm ( a , g ); 50 μm ( e ). Figure 3—source data 1. Percentages of branching modes, orthogonal bifurcations, and trifurcations in right accessor lung lobs of Svep1 wild type and mutant embryo at E14.5 . Figure 3—source data 2. Percentages of ectopic budding in right accessory lung lobes of Svep1 wild type and mutant embryos at E14.5 .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Tissue, Labeling, Knock-Out, Staining, Organ Culture, Two Tailed Test, Mutagenesis

    ( a ) Images of lung explants from E12.5 Svep1 +/+ embryos cultured for 48 hr. Images of lung explants from E12.5 Svep1 +/+ embryos cultured for 48 hr show a significant reduction in branching (control, top row) when treated with SVEP1 peptide (bottom row). ( b ) Plot showing peripheral airway counts of a left lung lobe after 48 hr cultured with and without SVEP1 treatment. This panel was created using BioRender.com . ( c ) Svep1 -/- lung explants treated with SVEP1 for 48 hr show a reduction in trifurcations compared to untreated lung explants. Black arrowheads indicate bifurcation; arrows indicate trifurcations (mean ± SEM; two-tailed unpaired t-test; control n=6, rSVEP1, n=7; p<0.05). Scale bars: 100 µm ( a, c ). Figure 4—source data 1. Peripheral airway fold change data for a left lung lobe explant following 48 hr cultured with and without SVEP1 treatment ( , panel b).

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Images of lung explants from E12.5 Svep1 +/+ embryos cultured for 48 hr. Images of lung explants from E12.5 Svep1 +/+ embryos cultured for 48 hr show a significant reduction in branching (control, top row) when treated with SVEP1 peptide (bottom row). ( b ) Plot showing peripheral airway counts of a left lung lobe after 48 hr cultured with and without SVEP1 treatment. This panel was created using BioRender.com . ( c ) Svep1 -/- lung explants treated with SVEP1 for 48 hr show a reduction in trifurcations compared to untreated lung explants. Black arrowheads indicate bifurcation; arrows indicate trifurcations (mean ± SEM; two-tailed unpaired t-test; control n=6, rSVEP1, n=7; p<0.05). Scale bars: 100 µm ( a, c ). Figure 4—source data 1. Peripheral airway fold change data for a left lung lobe explant following 48 hr cultured with and without SVEP1 treatment ( , panel b).

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Cell Culture, Control, Two Tailed Test

    ( a ) Interactome of downregulated genes in lungs of embryonic day 18.5 (E18.5) Svep1 -/- embryos with clusters labeled by enriched annotation terms. ( b ) Gene expression heatmap from RNA sequencing shows that significantly downregulated genes in lungs from Svep1 -/- embryos are associated with collagen and smooth muscle genes. ( c ) ACTA2 and calponin staining at E16.5. ( d ) ACTA2 and calponin localization in lung at E18.5 (white arrowheads indicate distal respiratory bronchioles; orange arrows indicate proximal bronchioles; white arrows indicate blood vessels). ( e ) ACTA2 staining (red) in wild-type and Svep1 -/- E12.5 lung explants, each cultured for 6 days, showed a reduction in smooth muscle cell formation in secondary and tertiary bronchi of the mutants (white arrows). ( f ) SOX2 immunofluorescence staining marking the progenitor cells of the epithelium in the proximal airways. Arrow marks the proximal airway at the core of the Svep1 -/- lung. ( g ) Immunofluorescence of the club cell marker SCGB1A1 (green) highlights the small proximal airways in mutant lungs. SCGB1A1 (green) co-localization with the ciliated cell marker FOXJ1 (pink) reveals a multicellular airway epithelium composed primarily of club cells. In Svep1 -/- embryos, the bronchiolar lumina are narrower than Svep1 +/+ (highlighted in white). Quantification shows an increased average of number of club cells (SCGB1A1) but not of ciliated cells (FOXJ1) (average counts ± SEM; t-test, p<0.05, n≤3). Scale bars: 200 µm or 100 µm. Figure 5—source data 1. Counts of SCGB1A1 and FOXJ1 positive cells in lungs of Svep1wild type and mutant embryos .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Interactome of downregulated genes in lungs of embryonic day 18.5 (E18.5) Svep1 -/- embryos with clusters labeled by enriched annotation terms. ( b ) Gene expression heatmap from RNA sequencing shows that significantly downregulated genes in lungs from Svep1 -/- embryos are associated with collagen and smooth muscle genes. ( c ) ACTA2 and calponin staining at E16.5. ( d ) ACTA2 and calponin localization in lung at E18.5 (white arrowheads indicate distal respiratory bronchioles; orange arrows indicate proximal bronchioles; white arrows indicate blood vessels). ( e ) ACTA2 staining (red) in wild-type and Svep1 -/- E12.5 lung explants, each cultured for 6 days, showed a reduction in smooth muscle cell formation in secondary and tertiary bronchi of the mutants (white arrows). ( f ) SOX2 immunofluorescence staining marking the progenitor cells of the epithelium in the proximal airways. Arrow marks the proximal airway at the core of the Svep1 -/- lung. ( g ) Immunofluorescence of the club cell marker SCGB1A1 (green) highlights the small proximal airways in mutant lungs. SCGB1A1 (green) co-localization with the ciliated cell marker FOXJ1 (pink) reveals a multicellular airway epithelium composed primarily of club cells. In Svep1 -/- embryos, the bronchiolar lumina are narrower than Svep1 +/+ (highlighted in white). Quantification shows an increased average of number of club cells (SCGB1A1) but not of ciliated cells (FOXJ1) (average counts ± SEM; t-test, p<0.05, n≤3). Scale bars: 200 µm or 100 µm. Figure 5—source data 1. Counts of SCGB1A1 and FOXJ1 positive cells in lungs of Svep1wild type and mutant embryos .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Labeling, Gene Expression, RNA Sequencing, Staining, Cell Culture, Immunofluorescence, Marker, Mutagenesis

    ( a ) In situ hybridization demonstrates increased expression of Fgfr2 in Svep1 -/- mutants. ( b ) Relative transcript abundance of Fgfr2 (and its isoforms Fgfr2b and Fgfr2c ), Fgf10 , Fgf9, and Kras showing increased expression in Svep1 -/- mutant embryos at embryonic day 18.5 (E18.5). ( c ) Svep1 +/+ and Svep1 -/- lung explants from E12.5 embryos treated ex vivo with the FGF signaling inhibitor SU5402 (2.5 µg/ml). Black arrows point to normal tip morphology in Svep1 +/+ lungs. The black arrowhead points to distal tip trifurcations that persist in lung explants from Svep1 -/- embryos. SOX9 (green) and ACTA2 (purple) staining confirm the branching anomalies (highlighted in the white dotted areas) in Svep1 -/- lung explants. White arrows mark normal bifurcations, white arrowheads indicate trifurcations and distal budding; the red arrowhead highlights ectopic budding not affected by the induction of FGF. ( d ) E12.5 lung explants treated with SU5402 (2.5 µg/ml) for 4 days demonstrate reduced ACTA2 localization in distal airways of Svep1 -/- lungs compared to wild-type lungs. ( e ) Fgf9 mRNA expression in wild-type (E14.5) and mutant lung (E16.5). ( f ) Wild-type E12.5 lung explants after 48 hr: untreated control (top left); treated with SVEP1 alone (top right); FGF9 alone (bottom left); combination of SVEP1 and FGF9 (bottom right). The combination restores a normal bud tip phenotype (average counts ± SEM; t-test, p<0.01 and <0.05, n>3). Scale bars: 100 µm ( a , c , d , e ). Figure 6—source data 1. Transcript abundance of Fgfr2, Fgfr2 isoforms, and Kras in Svep1-/- mutant embryos at E18.5 . Figure 6—source data 2. Lung bud tip growth fold change in treated and untreated Svep1 wild type E12.5 lung explants .

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) In situ hybridization demonstrates increased expression of Fgfr2 in Svep1 -/- mutants. ( b ) Relative transcript abundance of Fgfr2 (and its isoforms Fgfr2b and Fgfr2c ), Fgf10 , Fgf9, and Kras showing increased expression in Svep1 -/- mutant embryos at embryonic day 18.5 (E18.5). ( c ) Svep1 +/+ and Svep1 -/- lung explants from E12.5 embryos treated ex vivo with the FGF signaling inhibitor SU5402 (2.5 µg/ml). Black arrows point to normal tip morphology in Svep1 +/+ lungs. The black arrowhead points to distal tip trifurcations that persist in lung explants from Svep1 -/- embryos. SOX9 (green) and ACTA2 (purple) staining confirm the branching anomalies (highlighted in the white dotted areas) in Svep1 -/- lung explants. White arrows mark normal bifurcations, white arrowheads indicate trifurcations and distal budding; the red arrowhead highlights ectopic budding not affected by the induction of FGF. ( d ) E12.5 lung explants treated with SU5402 (2.5 µg/ml) for 4 days demonstrate reduced ACTA2 localization in distal airways of Svep1 -/- lungs compared to wild-type lungs. ( e ) Fgf9 mRNA expression in wild-type (E14.5) and mutant lung (E16.5). ( f ) Wild-type E12.5 lung explants after 48 hr: untreated control (top left); treated with SVEP1 alone (top right); FGF9 alone (bottom left); combination of SVEP1 and FGF9 (bottom right). The combination restores a normal bud tip phenotype (average counts ± SEM; t-test, p<0.01 and <0.05, n>3). Scale bars: 100 µm ( a , c , d , e ). Figure 6—source data 1. Transcript abundance of Fgfr2, Fgfr2 isoforms, and Kras in Svep1-/- mutant embryos at E18.5 . Figure 6—source data 2. Lung bud tip growth fold change in treated and untreated Svep1 wild type E12.5 lung explants .

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: In Situ Hybridization, Expressing, Mutagenesis, Ex Vivo, Morphology, Staining, Control

    ( a ) Expression heatmaps of selected genes associated with alveolar AT1 and AT2 cells show that Svep1 -/- lungs have lower expression of AT1 genes compared to Svep1 +/+ lungs and higher expression of AT2-associated genes. ( b ) Immunofluorescence of PDPN, HOPX, Pro-SFPC, and ABCA3 in the lungs of E18.5 Svep1 +/+ and Svep1 -/- embryos. Arrows indicate airway bud stalks. ( c ) PDGRFA+ (green) staining at E18.5. Plots of the average number of PDGFRA+ cells (average counts ± SEM; t-test, p<0.05, n=3). ( d ) Immunofluorescence of the mesothelial progenitor marker WT1 (red) with KI67 (green) staining indicating actively dividing WT1+ progenitors (white arrows). Plot of the number of WT1+ and WT+/KI67+ cells at E16.5 and E18.5. Svep1 -/- lungs have more WT1+ cells than Svep1 +/+ lungs at E18.5; however, the number of WT1+ KI67 lungs is unchanged between Svep1 +/+ and Svep1 -/- lungs. ( e ) Labeling of endothelial cells with CD31 illustrating the double layer of the saccular microvasculature in normal lungs (arrowhead) and disorganized microvasculature in Svep1 -/- lungs at E18.5. Scale bars: 250 µm ( b ); 100 µm ( c , d ); 5 µm ( e , h ). Scale bars: 25 µm ( c , e ); 50 µm ( b ); 100 µm ( c ). Figure 7—source data 1. Average counts of PDGFRA+ cells in Svep1 wild type ad mutant lungs at E18.5 ( , panel c). Figure 7—source data 2. Relative transcript abundance of WT1 in lungs of Svep1 wild type and mutant embryos at E18.5 . Figure 7—source data 3. Counts of WT1+ cells in lungs of Svep1 wild type and mutant embryos at E18.5 ( , panel d).

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Expression heatmaps of selected genes associated with alveolar AT1 and AT2 cells show that Svep1 -/- lungs have lower expression of AT1 genes compared to Svep1 +/+ lungs and higher expression of AT2-associated genes. ( b ) Immunofluorescence of PDPN, HOPX, Pro-SFPC, and ABCA3 in the lungs of E18.5 Svep1 +/+ and Svep1 -/- embryos. Arrows indicate airway bud stalks. ( c ) PDGRFA+ (green) staining at E18.5. Plots of the average number of PDGFRA+ cells (average counts ± SEM; t-test, p<0.05, n=3). ( d ) Immunofluorescence of the mesothelial progenitor marker WT1 (red) with KI67 (green) staining indicating actively dividing WT1+ progenitors (white arrows). Plot of the number of WT1+ and WT+/KI67+ cells at E16.5 and E18.5. Svep1 -/- lungs have more WT1+ cells than Svep1 +/+ lungs at E18.5; however, the number of WT1+ KI67 lungs is unchanged between Svep1 +/+ and Svep1 -/- lungs. ( e ) Labeling of endothelial cells with CD31 illustrating the double layer of the saccular microvasculature in normal lungs (arrowhead) and disorganized microvasculature in Svep1 -/- lungs at E18.5. Scale bars: 250 µm ( b ); 100 µm ( c , d ); 5 µm ( e , h ). Scale bars: 25 µm ( c , e ); 50 µm ( b ); 100 µm ( c ). Figure 7—source data 1. Average counts of PDGFRA+ cells in Svep1 wild type ad mutant lungs at E18.5 ( , panel c). Figure 7—source data 2. Relative transcript abundance of WT1 in lungs of Svep1 wild type and mutant embryos at E18.5 . Figure 7—source data 3. Counts of WT1+ cells in lungs of Svep1 wild type and mutant embryos at E18.5 ( , panel d).

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Expressing, Immunofluorescence, Staining, Marker, Labeling, Mutagenesis

    ( a ) Epithelial localization of surfactant-associated protein C (SFTPC; green) and SRY-box 9 (SOX9; purple) expression in the distal airway tips at E18.5. Arrows indicate the co-localization of SFPC and SOX9. ( b ) Perilipin 2 (PLIN2, aka ADFP) marks lipofibroblasts in the lung mesenchyme. Expression of the Plin2 (Adfp) gene in the lungs of Svep1 mutant and wild-type E18.5 embryos was not statistically different. Scale bars: 100 µm.

    Journal: eLife

    Article Title: Svep1 orchestrates distal airway patterning and alveolar differentiation in murine lung development

    doi: 10.7554/eLife.100443

    Figure Lengend Snippet: ( a ) Epithelial localization of surfactant-associated protein C (SFTPC; green) and SRY-box 9 (SOX9; purple) expression in the distal airway tips at E18.5. Arrows indicate the co-localization of SFPC and SOX9. ( b ) Perilipin 2 (PLIN2, aka ADFP) marks lipofibroblasts in the lung mesenchyme. Expression of the Plin2 (Adfp) gene in the lungs of Svep1 mutant and wild-type E18.5 embryos was not statistically different. Scale bars: 100 µm.

    Article Snippet: Lung explants from E12.5 or E13.5 Svep1 +/+ and Svep1 -/- embryos were treated for 2 days with a 146 amino acid recombinant protein antigen of Svep1 (10 μg/ml, NP_0735725.2, region 874–971, Novus Biologicals), for 2–4 days with and without an FGFR2 kinase inhibitor (2.5 μM SU5401, Pfizer) signaling and for 2 days with FGF9 (200 μM; R&D Systems) or FGF9+SVEP1 recombinant protein.

    Techniques: Expressing, Mutagenesis