Fig. 4.

Dual-input control of β-catenin and Lef1 levels in mESCs. a, left panel, f, g Dual-input regulation system consisting of the reverse transactivator (rtTA) and the conditionally destabilised β-catenin^S33Y (tagged or not with mCherry, a left panel and f, respectively), or Lef1 (g). a, right panel Representative flow cytometry profile of Doxy and/or TMP treated EF1a-rtTA_TRE3G-DDmCherryβ-catenin^S33Y mESCs. b DDmCherryβ-catenin^S33Y protein levels measured by western blot in EF1a-rtTA_TRE3G-DDmCherryβ-catenin^S33Y mESCs treated with Doxy and/or TMP; mCherry (upper panel) and β-catenin (lower panel) antibodies were used. Asterisk indicates a non-specific band. c, d β-catenin immunostaining in EF1a-rtTA_TRE3G-DDmCherryβ-catenin^S33Y mESCs treated for 24 h with TMP (c) or Doxy/TMP (d), using mCherry (red signal) and β-catenin (green signal) antibodies. DAPI was used to stain the nuclei. Zoomed pictures of selected clones are shown. Scale bars 25 μm. a–d Doxy and TMP were used at 1000 ng/mL and 100 nM, respectively. e Set-point relay control experiment, using inducers (Doxy 100 ng/mL and TMP 10 nM) as control inputs. In red the measured output (normalised mCherry fluorescence), in blue the reference fluorescence, set at 50% of the average values measured during the calibration phase (120 mins with continuous Doxy/TMP administration). The control performance indexes are reported in Supplementary Table 3 and Note 2; details about feedback control implementation are in Supplementary Note 2. h, i DDβ-catenin^S33Y (h) and DDLef1 (i) protein levels measured by western blot in EF1a-rtTA_TRE3G-DDβ-catenin^S33Y (h) and EF1a-rtTA_TRE3G-DDLef1 (i) mESCs following 24 h of treatment with increasing concentration of Doxy (0, 1, 5, 50, 100, 500, 1000 ng/mL) and maximum TMP (10μM), or increasing concentration of TMP (0, 1, 10, 100 nM; 1, 10 μM) and maximum Doxy (1000 ng/mL). Samples induced with the maximal concentration of inducers (Doxy1000ng/mL and TMP10μM) were loaded only once. j, k Dynamic response of EF1a-rtTA_TRE3G-DDβ-catenin^S33Y (j) and EF1a-rtTA_TRE3G-DDLef1 (k) mESCs to Doxy/TMP in a time-course experiment of 38 h, in which inducers were washed out after 14 h incubation. The concentration of inducer molecules used are Doxy 100 ng/mL and TMP 10 nM (j) or Doxy 1000 ng/mL and TMP 100 nM (k). Source data are provided as a Source Data file