Fig 2: Detection and quantification of PPIs at a single cell level by PLIC. a A graphical scheme illustrating the main steps of the PLIC protocol. FS fluorescent signal. b A graphical scheme illustrating PLIC analysis of Aire and Sirt1 interaction. c PLIC analysis showing representative images obtained from imaging flow cytometry of the interaction of Aire and Sirt1 in mTEChi and CD45+ populations presented as (left to right): bright field (BF), PLA signal (green), staining of CD80 (PB), EpCAM (APC), CD45 (APC/Cy7), and BF/PLA/EpCAM overlay (merged). d Quantitative analysis of Aire and Sirt1 interaction (data are presented as percentage of positive cells (PLA+) in mTEChi or CD45+ cells isolated from WT mice, compared to probes background. Shown are values from three independent experiments. Each point on the graph (triangle for mTEChi, circle for CD45+, and cross for probes only) represents an averaged value of single independent experiment, calculated from 2 to 4 biological replicates, +s.e.m). e PLIC analysis showing representative images of the interaction of Aire and Sirt1 in mTEChi overlaid with Aire staining (left to right): bright field (BF), Aire-AF488 (5H12), PLA signal (FarRed), BF/Aire/PLA/EpCAM overlay (merged), staining of EpCAM (APC/Cy7), CD80 (PB), and CD45 (PE/Cy7). Scale bar 7 µM

Fig 3: Loss of eNOS leads to sustained lung fibrosis in young animals following bleomycin challenge. (a) Nos3 transcriptional analysis of FACS-sorted CD31+/GFP-/CD45-/EpCAM- lung ECs isolated from young and aged mice after bleomycin-induced lung injury (young sham, N = 6; young 30 days, N = 10; young 75 days, N = 8; aged sham, N = 7; aged 30 days, N = 9; aged 75 days, N = 7). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one-way analysis of variance (followed by Tukey's post hoc test). (b) Lung homogenates from WT and eNOS-/- mice were analyzed via Western blot using anti eNOS and anti GAPDH antibodies. (c) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (WT sham, N = 7; WT 11 days, N = 3; WT 60 days, N = 10; eNOS-/- sham, N = 7; eNOS-/- 11 days, N = 3; eNOS-/- 60 days, N = 14). Data passed Shapiro–Wilk normality test, are expressed as mean ± SD, and analyzed using one-way analysis of variance (followed by Tukey's post hoc test). (d) Masson's trichrome assay was used to stain lung tissue. (e) Transcriptional analysis of whole lung homogenates obtained from WT and eNOS-/- mice (WT 60 days, N = 7; eNOS-/- 60 days, N = 4). The reference group throughout all the genes analyzed in this panel is WT 60 days after bleomycin. Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using Student's t test (*p < 0.05; **p < 0.01).

Fig 4: PLIC quantitative analysis of protein post-translational modification in mTECs. a A graphical scheme illustrating PLIC analysis of acetylated Aire, using mouse anti-Aire #209 antibody, and rabbit anti-lysine acetylation antibody. b PLIC analysis showing representative images of Aire acetylation in mTEChi and CD45+ cells isolated from Sirt1 +/– and Sirt1 –/– mice. Presented as (left to right): bright field (BF), PLA (green), staining of CD80 (PB), EpCAM (APC), CD45 (APC/Cy7), and BF/PLA/EpCAM overlay (merged). Scale bar 7 µM. c Quantitative analysis of Aire acetylation (presented as the percentage of PLA+ cells) in mTEChi or CD45+ cells isolated from Sirt1 +/– and Sirt1 –/– mice. Shown are values from three independent experiments. Each point on the graph (triangle for mTEChi, circle for CD45+, and cross for probes only) represents an average value of single independent experiment, calculated from 2 to 4 biological replicates, +s.e.m; **p-value = 0.0014 (independent student t-test)

Fig 5: SHP-2 interacts with CEACAM1 and blocks EMT in G-2 cells(A) Co-immunoprecipitation of SHP-2 with CEACAM1 in CEACAM1-expressing G-2scr cells, but not in G-2shCC1#3 with reduced CEACAM1 levels, demonstrates a physical interaction between SHP-2 and CEACAM1. (B) assessment of ß-catenin phosphorylation by Western blot in cells without (mock) or with SHP-2 inhibitor treatment (NSC87877, 100 µM, 24 hrs): treated cells display moderately increased levels of ß-catenin phosphorylation at Y86 as well as reduced ß-catenin phosphorylation at S33/S37/T41; concomitantly, the expression level of Vimentin is increased; quantities were normalized relative to ß-catenin levels. (C) Phase contrast images of CEACAM1-expressing G-2 cells treated with 100 µM SHP- 2 inhibitor NSC-87877 for 24 hrs or 72 hrs shows that mesenchymal transformation of the G-2 cells increases with time in comparison to mock treated cells; epithelial cell clusters are indicated by white dotted lines, mesenchymal cells with poor intercellular contacts and elongated cell bodies are indicated by black arrows; scale bar: 100 µm. (D) RT-qPCR-based gene expression analyses of EMT-signature marker genes in G-2 cells with (shaded bars) or without (white bars) SHP-2 inhibitor treatment (NSC-87877, 100 µM, 72 hrs). Note that the expression of Epcam and E-cadherin is significantly reduced cells with SHP-2 inhibition, whereas EMT drivers such as vimentin, Snai1, Twist1, Zeb1, Tgf1i1, and Wisp1 are elevated; Twist2 did not exhibit significant alteration of expression.