Fig 2: Piezo1 regulates IL-12 and TGFß1 production by dendritic cells (DCs) to direct TH1 and Treg cell differentiation.(A–B) Intracellular staining of IL-12p40 and TGFß1 expression in WT and Piezo1-/- splenic DCs after 5 hr of treatment with lipopolysaccharide (LPS) (A; 10 ng/ml) or culture on 2 and 50 kPa hydrogels (B). A representative figure shown on the left, and the data summarized on the right. (C) Intracellular staining of IFN? and Foxp3 in T cells cocultured with WT and Piezo1-/- splenic DCs in the presence of the indicated treatments (IL-12, Peprotech, 10 µg/ml or anti-TGFß1, R&D Systems, 20 µg/ml) for 5 days. A representative figure shown on the left, and the data summarized on the right. (D) Intracellular staining of IFN? (upper panel) and Foxp3 (lower panel) in T cells cocultured with WT and Piezo1?DC splenic DCs conditioned with 50 kPa hydrogel and the indicated treatments for 5 days and data summarized. The data are representative of three independent experiments (mean ± s.d.; n=3–5). ***p<0.001, compared with the indicated groups.

Fig 3: Piezo1 regulates TGFß1 and IL-12 production through the SIRT1-HIF1a-glycolysis pathway.(A–B) Sorted splenic dendritic cells (DCs) from WT or Piezo1-/- mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml; A) or with 2 or 50 kPa hydrogels (B) for 24 hr in the presence or absence of 2-deoxy-D-glucose (2-DG) (1 mmol/l). The proton production rate (PPR) was analyzed as a readout for glycolysis. (C) mRNA expression of glycolytic molecules in splenic DCs from WT or Piezo1-/- mice treated with LPS (10 ng/ml) for 12 hr. The levels in the WT control group were set to 1. (D) Intracellular staining of p38, Erk, and JNK phosphorylation and SIRT1 and HIF1a expression in splenic DCs from WT or Piezo1-/- mice. A representative figure shown in the upper panel, and the data summarized in the lower panel. (E) Splenic DCs from WT, Piezo1-/- , Sirt1-/-, and Piezo1/Sirt1 double knockout (DKO; Piezo1-/-Sirt1-/-) mice were stimulated with LPS (10 ng/ml). Intracellular staining of IL-12p40. A representative figure shown on the left, and the data summarized on the right. (F) Splenic DCs from WT or Hif1a-/- mice were stimulated with LPS (10 ng/ml) in the presence or absence of Yoda1 (25 µM). Intracellular staining of IL-12p40. A representative figure shown on the left, and the data summarized on the right. The data are representative of three independent experiments (mean ± s.d.; n=3–4). **p<0.01 and ***p<0.001, compared with the indicated groups.

Fig 4: Piezo1 regulates TGFß1 and IL-12 production through the calcium-calcineurin-NFAT axis.(A) Measurement of intracellular Ca2+ concentrations with Fura2 dye in splenic dendritic cells (DCs) from WT or Piezo1-/- mice treated with lipopolysaccharide (LPS) (10 ng/ml) or cultured on plates containing 50 kPa hydrogels. (B) Intracellular Ca2+ concentrations measured with Fura2 in splenic DCs from WT mice after the indicated treatment (Yoda1, 25 µM, MCE; ruthenium red, 30 µM, Sigma; gadolinium chloride, 10 µM, Sigma). (C–D) Intracellular staining of TGFß1 (C) and IL-12p40 (D) in splenic DCs from WT mice after the indicated treatments. (E) Intracellular staining of IL-12p40 in splenic DCs from WT mice after the indicated treatments. A representative figure shown on the left, and the data summarized on the right. (F) Intracellular staining of NFATc1 in splenic DCs from WT mice after the indicated treatments (CsA, 10 nM). A representative figure shown on the left, and data summarized on the right. (G–H) Intracellular staining of IL-12p40 in splenic DCs from WT or Piezo1-/- mice after the indicated treatments (Yoda1, 25 µM, MCE; 11R-VIVIT, 100 nM, MCE; CsA, 10 nM, Sigma). A representative figure shown on the left, and data summarized on the right. The data are representative of three to four independent experiments (mean ± s.d.; n=3–4). ***p<0.001, compared with the indicated groups.

Fig 5: vvDD-IL-23 treatment prolongs viral persistence. B6 mice were i.p. inoculated with 5×105 MC38-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×108 PFU/mouse five days after tumor inoculation. Tumor-bearing mice were sacrificed five or nine days post-treatment and primary tumors were collected and analyzed using RT-qPCR to determine the expression of A34R (A), IL12p40 (B), IL-10 (C) and IL-23R (D). The IL-23R expression on CD45- or CD45+ cells were determined by flow cytometry (E, F). MC38-luc (3×105 cells), CT26 (3×105 cells), B16 (2×105 cells), LLC (3×105 cells), EMT (3×105 cells), or AB12-luc (3×105 cells) tumor cells were mock-infected or infected with vvDD at an MOI of 1. The cell pellets were harvested to measure IL-23R expression at 24 h after infection using flow cytometry (G). MC38-luc-bearing mice treated as above were also sacrifice at D11, D13 and D17 to monitor viral persistence in tumors using RT-qPCR (H). *: P<0.05; **: P<0.01; and ****: P<0.0001. ns: not significant.