Figure 8. PRRX1 ASO attenuates lung fibrosis in mouse and Human Precision-cut Lung slices (PCLS).

(A) Before-after plots showing the mRNA expression of Prrx1a, Prrx1b, Acta2, Col1a1, and Fn1 (n=6) relative Control ASO alone condition (red dashed line) in mouse PCLS stimulated with fibrosis cytokine cocktail (FC) and then treated either with control ASO or PRRX1 ASO. (B) Representative immunoblot showing FN1, COL1, and ACTA2 expression (n=5) relative to control ASO alone condition (red dashed line) in mouse PCLS stimulated with FC and then treated either with control or Prrx1 ASO. The corresponding quantifications of ACTA2, COL1, and FN1 expression ratio to Tubulin are displayed as before-after plots on the right. Note that COL1 expression was decreased in 4 out 5 experiments. (C) Before-after plots showing the mRNA expression of PRRX1a, PRRX1b, ACTA2, COL1A1 and FN1 (n=3) relative Control ASO condition (red dashed line) in Human PCLS treated either with control ASO or PRRX1 ASO in presence or absence of FC. COL1A1 upregulation was lessened in 2 out 3 experiments while ACTA2 and FN1 levels were decreased in 3 out of 3 experiments. (D) Representative picrosirius staining (n=3) in Human PCLS treated with control ASO alone (left panel, basal condition) or after stimulation with Fibrosis Cytokine cocktail and treated with either control (middle panel) or PRRX1 (right panel) ASO. Nuclei were counterstained with hematoxylin. The quantification of picrosirius staining relative to control ASO alone (red dashed line) is showed on the right (Before-after plot). Scale bar: 50 µm. Abbreviations: Precision-Cut Lung slices (PCLS), Fibrosis Cytokine Cocktail (FC), Control (Cont), Antisense oligonucleotide (ASO), Stimulation (stim.). Wilcoxon test * P≤0.05.

Figure 8—figure supplement 1. effect of PRRX1 ASO on the expression of myofibroblastic differentiation markers in mouse and Human PCLS at basal condition.

(A) Dot plots with median showing the relative mRNA expression of Prrx1a, Prrx1b, Acta2, Col1a1, and Fn1 in mouse PLCS treated with Prrx1 ASO alone (n=8) relative to control ASO (red dashed line). Note that Prrx1a, Prrx1b, and Acta2 mRNA levels were decreased in presence of Prrx1 ASO compared to control ASO. (B) Immunoblot showing FN1, COL1 and ACTA2 expression in mouse PCLS (n=6) treated with control or Prrx1 ASO. TUB was used as loading control. Right part: quantification of COL1, FN1 and ACTA2 relative expression to TUB in mouse PCLS treated with Prrx1 ASO relative to control ASO (red dashed line). (C) Dot plots with median showing the relative mRNA expression of PRRX1A, PRRX1B, ACTA2, COL1A1, and FN1 in control Human PLCS (n=5) treated with PRRX1 ASO alone relative to control ASO (red dashed line). Note that PRRX1a, PRRX1b mRNA levels were effectively downregulated in presence of PRRX1 ASO compared to control ASO. (D) Column with median and individual values showing the relative mRNA expression of PRRX1A, PRRX1B, ACTA2, A1COL1, and FN1 in Human IPF PLCS (n=3) treated with PRRX1 ASO alone relative to control ASO (red dashed line). Each color represents individual experiments. Abbreviations: Precision-Cut Lung slices (PCLS), Control (Cont), Antisense oligonucleotide (ASO). Wilcoxon signed-rank test, $ p≤0.05 $$ p≤0.01.