Extended Data Fig. 4: Kinetics and sufficiency of cyclical hydrostatic pressure induced HIF1α stabilization and transcriptional response.

a, Representative immunoblot analysis of HIF1α and β-tubulin from BMDMs in CHP with 5 μM GsMTx4 (Gsm) for 6 h, then washed three times in DMEM before further CHP stimulation. Data are representative of two independent experiments. b, Immunoblot analysis of HIF1α and β-tubulin from BMDMs in CHP for 6 h, pretreated with 10 μM BAPTA-AM for 30 min. Data are representative of two independent experiments. c, Immunoblot analysis of HIF1α and β-tubulin in BMDMs. Piezo1^fl/fl (WT) or Piezo1^ΔLysM (KO) were cultured in CHP for 6 h along with 3 μg ml BFA. Cells were then washed with DMEM three times and further subjected to additional CHP stimulation. Supernatant (sup) was then clarified and transferred to WT or KO BMDMs cultured in static pressure for two additional hours. Data are representative of two independent experiments. d, Representative immunoblot analysis of HIF1α and β-tubulin from BMDMs treated with 100 μM chloroquine (ChlQ) or 50 μM MG132 for 6 h with CHP. Data are representative of two independent experiments. e, RT–qPCR analysis of Edn1 from Hif1a^fl/fl and Piezo1^ΔLysM BMDMs treated with CHP for 1 h or 6 h. Data are presented as three biological replicates. f, g, RT–qPCR analysis of Piezo1^fl/fl (f) or Piezo1^ΔLysM (g) BMDMs cultured in 6 h of treatment 200 μM DMOG, 6 h of CHP, 2 h of CHP followed by 4 h of no treatment (NT), 2 h of static pressure followed by 4 h of 200 μM DMOG, or 2 h CHP followed by 4 h of 200 μM DMOG. Data are representative of three biological replicates. Data are mean ± s.e.m.