(A) Co-staining of SOX9 (green) for distal buds, SOX2 (blue) for proximal epithelium, and SOX21 (red) at different stages of lung development. Boxed areas are sown as enlarged inserts. Schematic …
Quantification of the number of cells represented as Dorsal-Ventral ratio of SOX2 and SOX21 positive cells.
(A) Immunostaining on sections of the main bronchi immediately distal of the carina from mice at embryonic day (E) 12.5, 13.5, 14.5, and 15.5 using SOX2 (blue), SOX21 (red), and either SOX9 (green, …
(A) Immunofluorescence of SOX2 (blue), SOX21 (red), and TRP63 (green) on lung sections of E12.5, E13.5, and E14.5 showing a spatial co-localization of the three proteins. Scale bar = 100 µm. (B) …
Quantification of the number of SCGB1A1, FOXJ1 and TRP63 positive cells represented in the plots.
(A) Immunofluorescence of SOX2 (blue), SOX21 (red), and TRP63 (green) on lung sections of embryonic ages (E) 11.5. T = thymus, L = lung, and E = esophagus. Scale bar = 100 µm. (B) Immunofluorescence …
(A–D) Immunofluorescence and quantification of the number of TRP63+ basal cells (A), TRP73+ cells (B), FOXJ1+-ciliated cells (C), and KI67+-dividing cells (D) at E14.5 in wild-type (WT), Sox21+/−, …
Quantification of the number of positive cells represented in the plots.
(A–D) Immunofluorescence and quantification of the number of TRP63+ basal cells (A), TRP73+ cells (B), FOXJ1+-ciliated cells (C), and KI67+-dividing cells (D) at E14.5 in wild-type (WT) and Sox2+/− m…
(A) Schematic overview of mouse tracheal epithelial cell (MTEC) culture. QPCR analysis of Sox2 and Sox21 expression during differentiation of MTECs on air–liquid interface (ALI). One-way ANOVA (n = 5…
Quantification of the number of positive cells represented in the plots.
(A) Schematic overview of mouse tracheal epithelial cell (MTEC) culture experiment. QPCR analysis of TRP63, SCGB3A2 and FOXJ1 expression during differentiation of MTECs on air–liquid interface …
(A) Immunofluorescence staining on tracheal sections of adult wild-type mice for SOX2 (red), SOX21 (green), and KRT5 (grey, top row) or smooth muscle actin (grey, SMA, bottom row). TE = tracheal …
Quantification of the number of FOXJ1, SCGB1A1, TRP63 and KI67 positive cells represented in the plots.
(A) Immunofluorescence staining on tracheal sections of wild-type (WT), Sox2+/−, and Sox21+/−, 2 days post injury (2 DPI) of Keratin 5 (KRT, grey), Keratin 8 (KRT8, green), TubilinIV (TUBIV, red). (B…
(A) Immunofluorescence on human fetal lung sections post-conceptional week (PCW) 13, 16.5, and 17. Proximal–distal patterning of human developing airways is analyzed with SOX9 (red) and SOX2 (blue) …
(A) Schematic overview of human primary bronchial epithelial cell (HPBEC) culture. QPCR analysis of SOX2 and SOX21 expression during differentiation of HPBECs on air–liquid Interface (ALI). One-way …
Quantification of the number of positive cells represented in the plots.
(A) Bright field images of human fetal lung tip organoids (scale bar = 250 µm). (B) QPCR analysis of TP63, SCGB3A1, and FOXJ1 expression during differentiation of HPEBCs on air–liquid Interface …
5 lungs of WT, Sox2Sox2+/-, Sox21+/-, Sox21-/- and Sox2Sox2+/-Sox21+/- mice.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (M. musculus) | Sox2-CreERT | PMID:21982232 | MGI:5295990 | Sox2tm1(cre/ERT2)Hoch The Jackson Laboratories Stock No: 017593 |
Genetic reagent (M. musculus) | Sox21-KO | PMID:19470461 | MGI:2654070 | Dr. Stavros Malas |
Genetic reagent (M. musculus) | iSox2 | PMID:18374910 | MGI:98364 | Dr. Robbert Rottier |
Genetic reagent (M. musculus) | iSox21 | - | MGI:2654070 | Dr. Robbert Rottier |
Genetic reagent (M. musculus) | SPC-rtTA | PMID:11874100 | MGI:109517 | Prof. Jeffrey Whitsett |
Genetic reagent (M. musculus) | CC10-rtTA | PMID:10766812 | MGI:98919 | Prof. Jeffrey Whitsett |
Antibody | Mouse anti-β-TUBULIN IV | BioGenex | MU178-UC | 1:100 |
Antibody | Mouse anti-FOXJ1 | eBioscience | 14–9965 | 1:200 |
Antibody | Rabbit anti-KRT5 | Biolegend | Poly19055 | 1:500 |
Antibody | Rat anti-KRT8 | DSHB | TROMA-I | 1:100 |
Antibody | Rabbit anti-MYC (Immunofluorescence) | Abcam | AB9106 | 1:500 |
Antibody | Rabbit anti-SCGB1A1 | Abcam | AB40873 | 1:200 |
Antibody | Mouse anti-SCGB3A1 | R and D systems | AF1790 | 1:200 |
Antibody | Goat anti-SCGB3A2 | R and D systems | AF3465 | 1:500 |
Antibody | Mouse anti-SMA | Neomarkers | MS-113-P1 | 1:500 |
Antibody | Rabbit anti-SOX2 | Seven-Hills | WRAB-SOX2 | 1:500 |
Antibody | Goat anti-SOX2 | Immune systems | GT15098 | 1:500 |
Antibody | Goat anti-SOX21 | R and D systems | AF3538 | 1:100 (TSA: 9 min) |
Antibody | Rabbit anti-SOX9 | Abcam | AB185230 | 1:500 |
Antibody | Mouse anti-TRP63 | Abcam | AB735 | 1:100 |
Antibody | Rabbit anti-TRP73 | Abcam | AB40658 | 1:100 |
Antibody | Rabbit anti-FLAG | Sigma | F7425 | Western blot, 1:1000 |
Antibody | Rabbit anti-MYC | Abcam | AB9106 | 1:1000 |
Antibody | Rabbit anti-MUC5B | Sigma | HPA008246 | 1:500 |
Antibody | Mouse anti-β -TUBULIN | Sigma | T8328 | 1:2000 |
Antibody | Alexa Fluor 405, 488, 594 Donkey anti Goat IgG | Jackson ImmunoResearch | 705-475-147, 705-545-147, 705-585-147 | 1:500 |
Antibody | Alexa Fluor 488, 594, 647 Donkey anti Mouse IgG | Jackson ImmunoResearch | 715-545-151, 715-585-151, 711-605-151 | 1:500 |
Antibody | Alexa Fluor 488, 594, 647 Donkey anti Rabbit IgG | Jackson ImmunoResearch | 711-545-152, 711-585-151, 711-605-152 | 1:500 |
Antibody | Alexa Fluor 488, 594 Donkey anti Rat IgG | Jackson ImmunoResearch | 712-545-150 712-585-153 | 1:500 |
Antibody | Donkey anti-Goat HRP conjugated | Jackson ImmunoResearch | 705-035-147 | 1:500 |
KSFM-hPBEC medium | |||
---|---|---|---|
Reagent | Company | Cat. no. | Final concentration |
KSFM | Gibco | 17005034 | n/a |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
Bovine pituitary extract | Gibco | 13028014 | 0.03 mg/ml |
Human EGF | Peprotech | 315–09 | 25 ng/ml |
Isoproterenol | Sigma | I-6504 | 1 µM |
KSFM-MTEC expansion medium | |||
Reagent | Company | Cat. no. | Final Concentration |
KSFM | Gibco | 17005034 | n/a |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
Bovine pituitary extract | Gibco | 13028014 | 0.03 mg/ml |
Mouse EGF | Peprotech | 315–09 | 25 ng/ml |
Isoproterenol | Sigma | I-6504 | 1 µM |
Rock Inhibitor (Y27632) | Axon MedChem | 1683 | 10 µM |
DAPT | Axon MedChem | 1484 | 5 µM |
MTEC proliferation medium | |||
Reagent | Company | Cat. no. | Final concentration |
DMEM:F12 | Gibco | 1133032 | n/a |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
NaHCO3 | Gibco | 25080094 | 0.03% (w/v) |
Fetal calf serum | HyClone | SH30071.03 | 5% |
L-Glutamine | Gibco | 25030081 | 1.5 mM |
Insulin-transferin-selenium | Gibco | 41400045 | 1× |
Cholera toxin | Sigma | C8052 | 0.1 µg/ml |
Bovine pituitary extract | Gibco | 13028014 | 0.03 mg/ml |
Mouse EGF | Peprotech | 315–09 | 25 ng/ml |
Rock inhibitor (Y27632) | Axon MedChem | 1683 | 10 µM |
Retinoic acid | Sigma | R2625 | 0.05 µM |
MTEC differentiation medium | |||
Reagent | Company | Cat. no. | Final concentration |
DMEM:F12 | Gibco | 1133032 | n/a |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
NaHCO3 | Gibco | 25080094 | 0.03% (w/v) |
Bovine serum albumin | Gibco | 15260037 | 0.1% (w/v) |
L-Glutamine | Gibco | 25030081 | 1.5 mM |
Insulin-transferin-selenium | Gibco | 41400045 | 1× |
Cholera toxin | Sigma | C8052 | 0.025 µg/ml |
Bovine pituitary extract | Gibco | 13028014 | 0.03 mg/ml |
Mouse EGF | Peprotech | 315–09 | 5 ng/ml |
Retinoic acid | Sigma | R2625 | 0.05 µM |
Human fetal organoid medium | |||
Reagent | Company | Cat. no. | Final concentration |
Advanced DMEM:F12 | Invitrogen | 12634–034 | n/a |
R-Spondin | Peprotech | 120–38 | 500 ng/ml |
Noggin | Peprotech | 120–10C | 100 ng/ml |
Fgf10 | Peprotech | 100–26 | 100 ng/ml |
Fgf7 | Peprotech | 100–19 | 100 ng/ml |
EGF | Peprotech | AF-100–15 | 50 ng/ml |
CHIR99021 | Stem Cell Techn. | 72052 | 3 µM |
SB431542 | Tocris | 1614 | 10 µM |
B27 supplement (- VitA) | ThermoFisher | 12587–010 | 1× |
N-Acetylcysteine | Sigma | A9165 | 1.25 mM |
Glutamax 100× | Invitrogen | 12634–034 | 1× |
N2 | ThermoFisher | 17502–048 | 1× |
Hepes | Gibco | 15630–56 | 10 mM |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
Primocin | Invivogen | Ant-pm-1 | 50 µg/ml |
Human airway organoid medium | |||
Reagent | Company | Cat. no. | Final concentration |
Advanced DMEM:F12 | Invitrogen | 12634–034 | n/a |
R-Spondin | Peprotech | 120–38 | 500 ng/ml |
Noggin | Peprotech | 120–10C | 100 ng/ml |
Fgf10 | Peprotech | 100–26 | 100 ng/ml |
Fgf7 | Peprotech | 100–19 | 25 ng/ml |
SB202190 | Sigma | S7067 | 500 nM |
A83-01 | Tocris | 2939 | 500 nM |
Y-27632 | Axon MedChem | 1683 | 5 µM |
B27 supplement | Gibco | 17504–44 | 1× |
N-Acetylcysteine | Sigma | A9165 | 1.25 mM |
Nicotinamide | Sigma | N0636 | 5 mM |
Glutamax 100× | Invitrogen | 12634–034 | 1× |
Hepes | Gibco | 15630–56 | 10 mM |
Penicillin/streptomycin | Lonza | DE17-602e | 100 U/ml 100 µg/ml |
Primocin | Invivogen | Ant-pm-1 | 50 µg/ml |
RT-primers | |||
---|---|---|---|
Gene | Forward (5’→3’) | Reverse (5’→3’) | Species |
Foxj1 | CAGACCCCACCTGGCAGAATTC | AAAGGCAGGGTGGATGTGGACT | Mouse |
Gapdh (housekeeping) | CCTGCCAAGTATGATGACAT | GTCCTCAGTGTAGCCCAAG | Mouse |
Krt5 | TACCAGACCAAGTATGAGGAG | TGGATCATTCGGTTCATCTCAG | Mouse |
Scgb1a1 | GCAGCTCAGCTTCTTCGGACA | TCCTGGTCTCTTGTGGGAGGG | Mouse |
Scgb3a2 | GTGGTTATTCTGCCACTGCCCTT | TCGTCCACACACTTCTTCAGTCC | Mouse |
Sox2 | AACATGGCAATCAAATGTC | TTGCCAGTACTTGCTCTCAT | Mouse |
Sox21 | TTGAAAGATGCCTCTCACCA | AATAAGCTAAATGGGAAGGGAG | Mouse |
Trp63 | GGAAAACAATGCCCAGACTC | GATGGAGAGAGGGCATCAAA | Mouse |
Actin (housekeeping) | ATTGGCAATGAGCGGTTC | GGATGCCACAGGACTCCAT | Human |
Foxj1 | CCCACCTGGCAGAATTCAATCCG | CAGTCGCCGCTTCTTGAAAGC | Human |
Scgb1a1 | GCTCCGCTTCTGCAGAGATCTG | GCTTTTGGGGGAGGGTGTCCA | Human |
Scgb3a1 | TGCTGGGGGCCCTGACA | ACGTTTATTGAGAGGGGCCGG | Human |
Sox2 | AATGCCTTCATGGTGTGGTC | TTGCTGATCTCCGAGTTGTG | Human |
Sox21 | CCACTCGCTTGGATTTCTGACACA | TCGACTCAAACTTAGGGCAACGA | Human |
Trp63 | CCACCTGGACGTATTCCACTG | TCGAATCAAATGACTAGGAGGGG | Human |
Primers used for pGL4.10[luc2] cloning | ||
---|---|---|
Name | Primers (5’→3’) cut site | Cut site |
Trp63 | Forward: CAGGGTACCGGGCACATTCCATCTTTCCT | KpnI |
Reverse: CAGCTCGAGAGACTGGTCAAGGCTGCTCT | Xho | |
Sox2 | Forward: CAGGGTACCCGCGAGAGTATTGCAGGGAA | KpnI |
Reverse: CAGGCTAGCCGGAGATCTGGCGGAGAATA | NheI | |
Trp73 | Forward: CAGGGTACCGGACACGCATCTGTTGTGGA | KpnI |
Reverse: CAGCTCGAGTCTGCACACGCTGAGGAGCT | Xho |