Fig 2: Clonal expansion requires loss of p53, and p53 regulates TLR3 expression in rat lung CD117+ ECs and EC clones and human pulmonary artery ECs from PAH patients(A and B) qRT-PCR data show lower Tp53 and Tlr3 expression after four generations of clonal expansion in CD117+ lin- CD31+ rat lung ECs.(C) Clonal expansion was vastly reduced in CD117+ rat lung ECs when treated for 14 days with Nutlin 3a, which inhibits p53 protein degradation. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group.(D) qRT-PCR shows reduced mRNA expression of Tp53, Tlr3, and Id1 following siRNA targeting p53 versus control (scrambled, scrm) siRNA in rat lung CD117+ ECs. n = 5–6.(E and F) Representative images (scale bar: 250 µm, stitched images) and quantification of total network area and network length from 2D matrigel tube formation assay in CD117+ rat lung ECs after treatment with siRNA targeting p53 or scrambled (scrm) unspecific siRNA. n = 4 per group.(G and H) Representative western blots (G) and quantification of three independent experiments (H) show p53 protein accumulation following treatment with the MDM2 inhibitor Nutlin 3a. n = 9 per group total.(I) qRT-PCR shows increased expression of Tlr3, Mdm2, and Id1 following Nutlin 3a treatment. n = 6 per group.(J and K) Representative images (scale bar: 250 µm, stitched images) and quantification of total network area and network length in rat lung CD117+ fourth generation EC clones after treatment with Nutlin 3a or vehicle. n = 5 per group.(L and N) Representative western blots of p53, TLR3, BMPR2, phospho-(p)-IRF3, and p-Smad1/5/9 (L) and densitometric quantification of p53, TLR3, and BMPR2 from 2 independent experiments (n = 3 each) in PAECs from PAH patients following Nutlin 3a or vehicle treatment for 24 h.(O) qRT-PCR shows increased TLR3 mRNA expression following Nutlin 3a treatment in PAH PAECs. n = 5 per group.(P) Quantification of total network area and network length in PAH PAECs after treatment with Nutlin 3a or vehicle. n = 10 per group. Quantification data are n-fold of the arithmetic mean of veh.(Q) Clonal expansion was vastly reduced in PAECs following 14 days of treatment with Nutlin 3a. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group. All values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (t test).See also Figures S2–S9.

Fig 3: Increased TLR3 signaling promotes BMPR2 signaling in p53-deficient PAECs(A and B) Representative histograms of 5-bromodesoxyuridine (BrdU) pulsed (4h) and BrdU-antibody-stained (AF647) PAECs treated with adenoviruses expressing sh-scrm (control sh RNA), sh-TP53, and sh-TP53 combined with 25 µg/mL Poly(I:C). (B) Quantification from 2 different cell lines (total n = 6 each). Data indicate increased DNA synthesis in sh-TP53 cells, which is reduced by Poly(I:C). Data are shown after normalization to the arithmetic mean of the sh-scrm group.(C and D) Representative phase contrast images (scale bar: 500 µm, stitched images, C) and quantification of total network area and network length (D) in rat lung CD117+ fourth generation EC clones after treatment with Nutlin 3a or vehicle. Two different cell lines (total n = 12 each). Data are shown after normalization to the arithmetic mean of the sh-scrm group.(E and F) Western blots (E) and densitometric quantification (F) of TLR3, p-IRF3, BMPR2, p-Smad1/5/9, and Smad1 in PAECs treated with sh-scrm, sh-TP53, and sh-TP53 + Poly(I:C). The results have been expressed as a ratio of the loading control ß-actin and normalized to the arithmetic mean of the sh-scrm group.(G) qRT-PCR quantification of steady-state mRNA levels of TP53, TLR3, CXCL10, BMP2, and BMP4. The results are expressed as a ratio of the housekeeping gene and normalized to the arithmetic mean of the sh-scrm group. The data are shown as single data points and mean ± SEM. Data are shown as single data points and mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA).