Fig. 2: Development and characterization of the CaDox system.
From: Tumour priming by ultrasound mechanogenetics for CAR T therapy
a, NFAT nuclear translocation following repeated FUS stimulations, visualized via EGFP-tagged NFAT. Scale bar, 10 µm. b, Schematic of the calcium-gated and doxycycline-gated genetic circuit (CaDox). CMV, cytomegalovirus promoter; GOI, gene of interest. c,d, Time courses (c) and representative images (d) of NFAT translocation with variants exhibiting different calcineurin affinities. Red and green arrows in a and d indicate nuclei showing NFAT translocation, whereas white arrows in d mark nuclei without detectable translocation. ATP (60 µM) was applied at 2 min and washed off at 7 min (n = 5 biological replicates). N/C, nuclear over cytoplasmic ratio; WT, wild type. e, Diagram of the CaDox circuit comprising the CaDox regulator and reporter modules. PGK, phosphoglycerate kinase promoter. f, CaDox regulator translocation in response to ionomycin (1 µM). g, Quantification of CaDox-mediated gene induction using FLuc as the inducible reporter and RLuc as the constitutive control (n = 3 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (two-sided). Adjusted P value is as follows: Dox versus Dox/Iono, P < 0.0001; Dox, doxycycline. Iono, ionomycin; NC, negative control. h, Dose–response curve of doxycycline concentration in the presence or absence of ionomycin (n = 3 per group). Statistical significance was assessed using two-way ANOVA with Šidák’s multiple comparisons test (two-sided). Adjusted P values are as follows: 100 nM, P = 0.0001; 1 µM, P < 0.0001; 10 µM, P < 0.0001. i,j, Representative images (i) and quantifications (j) of mCherry reporter expression in PC-3 cells under indicated conditions (n = 31). DIC, differential interference contrast. One-way ANOVA with Tukey’s multiple comparisons test (two-sided); adjusted P values are as follows: ATP versus Dox, P < 0.0001; Dox versus Dox/ATP, P < 0.0001. Scale bar, 100 µm. Data in g, h and j are represented as mean ± s.e.m. Biological replicates were defined as independent wells or dishes. Images in a, d, f and i are representative of ≥3 independent experiments. *** and **** denote adjusted P < 0.001 and P < 0.0001, respectively. NS, not significant. Panel b created with BioRender.com.
