Fig. 3.

RYK inhibits cell death in the lung mesenchyme. (A) RNA-sequencing fold change (log₂) of differentially expressed genes in P0 Dermo1-Ryk^WT (n = 3) compared with Dermo1-Ryk^cKO (n = 3) lungs. (B) qPCR analysis of Bcl3 and Ccl2 mRNA levels in P0, P1, and P2 Dermo1-Ryk^WT and Dermo1-Ryk^cKO lungs. (C) qPCR analysis of Casp4 and Fas mRNA levels in P0, P1, and P2 Dermo1-Ryk^WT and Dermo1-Ryk^cKO lungs. (D) Representative Western blot (from three individual sets of lung lysates) for CCL2 in P0, P1, and P2 Dermo1-Ryk^WT and Dermo1-Ryk^cKO lungs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Values represent the densitometric ratio after normalization to GAPDH. (E) Immunostaining for CCL2 in P2 Dermo1-Ryk^WT (n = 6) and Dermo1-Ryk^cKO (n = 6) lung sections. (F) Representative Western blot (from three individual sets of lung lysates) for cleaved CASP3 and CASP4 in P0, P1, and P2 Dermo1-Ryk^WT and Dermo1-Ryk^cKO lungs. Values represent the densitometric ratio after normalization to GAPDH. (G) Immunostaining for cleaved CASP3, α-SMA, and E-cadherin in P2 Dermo1-Ryk^WT (n = 6) and Dermo1-Ryk^cKO (n = 6) lung sections. (H) Representative Western blot (from three individual sets of cell lysates) for NF-κB, phospho–NF-κB, CCL2, cleaved CASP3, and CASP4 in nontransfected and Ryk siRNA (10 nM or 50 nM)-transfected NIH 3T3 fibroblasts. Values represent densitometric ratios after normalization to GAPDH, except for phospho–NF-κB, which was normalized to GAPDH and total NF-κB. (I) Representative Western blot (from three individual sets of cell lysates) for NF-κB, phospho–NF-κB, CCL2, and cleaved CASP3 upon stimulation with WNT-3A and/or cotransfection of Ryk siRNA in NIH 3T3 fibroblasts. Values represent densitometric ratios after normalization to GAPDH, except for phospho–NF-κB, which was normalized to GAPDH and total NF-κB. (J) TUNEL staining and immunostaining for α-SMA (to mark smooth muscle) and E-cadherin in DMSO- or Z-DEVD-FMK–treated P3 Dermo1-Ryk^WT (n = 6) and Dermo1-Ryk^cKO (n = 6) lung sections. Arrows point to TUNEL-positive cells. (K) Representative Western blot (from three individual sets of lung lysates) for NF-κB, phospho–NF-κB, and CCL2 in DMSO- or Z-DEVD-FMK–treated P3 Dermo1-Ryk^WT and Dermo1-Ryk^cKO lungs. Values represent densitometric ratios after normalization to GAPDH, except for phospho–NF-κB, which was normalized to GAPDH and total NF-κB. Error bars are means ± SEM, two-tailed Student’s t test. Ct values are listed in SI Appendix, Table S2. (Scale bars: 50 μm for G and 30 μm for E and J.)