Supplementary Figure 4: Conditional ablation of Piezo1 in FRCs.

(a) Transcriptional expression measured by RNAseq of Piezo1 in FRCs and HEV ECs isolated from the PP of control or PEG-treated mice. (n= 7 mice per group (FRC) or 4 mice per group (HEV EC)) (b) Validation of specific gene recombination in exons 20-23 of Piezo1. Genomic DNA isolated from PP stromal cells enriched by depletion of CD45+ cells using magnetic beads, primary FRCs expanded in culture for 5 days, or tail snips. Genotypes of mice indicated below each lane. Bands indicate the following: Intact gene flanked by flox sequences = 330 bp, knockout = 230bp, wildtype = 160bp. (c) Flow cytometric analysis of the distribution of eYFP-expressing cells isolated from the PP and inguinal LNs of Ccl19cre⁺ x ROSA26-eYFP mice. (n=3 mice per group). (d) Confocal microscopy of cultured PP FRCs. FRCs were enriched and culture expanded from Ccl19cre⁺ x ROSA26-eYFP mice. Representative of two independent experiments. (e) Transcriptional expression of Piezo1 in PP FRCs isolated from WT and Ccl19-P1cKO mice. FRCs were enriched and culture expanded before mRNA was measured by qPCR (n= 4 (Cre⁻) or 3 (Cre⁺) mice). Data represented as mean ± SEM. Groups were compared by unpaired two tailed Student’s t test. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.005). No statistical significance is indicated with ‘ns’.