Figure 1: Schematics of the PNFL motif and ‘switch-OFF’ experiments following pulses of doxycycline of different duration.

(a) The tetracycline-controlled transactivator (tTA) is self-regulated, in the absence of doxycycline, by binding the tTA-responsive CMV-TET promoter, thus generating a PFL (black lines), whose dynamics is tracked by a destabilized EYFP (d2EYFP). The same CMV-TET promoter drives the transcription of the human miRNA miR-223 embedded in the first intron of the low-affinity nerve growth factor receptor (ΔLNGFR)²³, followed by a reporter gene encoding for the mCherry fluorescent protein (NFL—red lines). miR-223 in turn downregulates the tTA mRNA levels through four-repeated target sequences perfectly complementary to the miR-223 seed sequence, placed at the 3′-untraslated region of the PFL gene expression cassette. Doxycycline interrupts the tTA-mediated transcriptional activation. WPRE, woodchuck hepatitis virus post-transcription regulatory element. (b) Simulated d2EYFP fluorescence of PNFL cells following simulated treatment with doxycycline of different duration Δ. In the inset, the bifurcation diagram when varying the miRNA strength (λ) is shown. (c,d) Experimental d2EYFP fluorescence using the microfluidics device (solid green line) following treatment with doxycycline (red line) at time 120 min and removal after Δ=60 min (c) or 240 min (d); s.d. is among at least three replicates (thin green lines); simulations (blue and purple lines) are rescaled to the experimental data and also represented in b (same colours).