Fig. 5: Ectopic PRDM14 expression or desensitisation to auxin rescue hPGCLC specification.
From: A critical role of PRDM14 in human primordial germ cell fate revealed by inducible degrons
a IAA and/or Shield-1 (Sh) were added on indicated days of differentiation to deplete the endogenous PRDM14 or stabilise the PRDM14-DD transgene, respectively. “Control” cell line lacks both the AID knock-in and the PRDM14-DD transgene. Data show mean ± SD for n = 4 (PRDM14-DD22) or mean of n = 2 (control) independent experiments. Statistical significance was analysed by two-way ANOVA followed by Sidak’s multiple comparison test; for PRDM14-DD22: D0 IAA no Sh vs. no IAA no Sh not significant, (P = 0.4159), D0 IAA no Sh vs. no IAA D0 Sh ***(P = 0.0001), D0 IAA no Sh vs. no IAA D1 Sh *(P = 0.0125), D0 IAA no Sh vs. D0 IAA D0 Sh ***(P = 0.0008), D0 IAA no Sh vs. D0 IAA D1 Sh not significant (P = 0.8662); for control: all not significant (P > 0.97). b Scheme of the double AID/JAZ degron design. Endogenous PRDM14 is under the control of the AID degron as above, while the stability of TIR1 hormone receptor is controllable by the JAZ degron. c AID/JAZ validation by IF in hESCs (representative of three experiments). Simultaneous IAA and Cor supplementation depletes TIR1 and restores PRDM14, as shown by IF staining for PRDM14-AID-Venus and TIR1(V5-tagged). Nuclei were counterstained by DAPI. Scale bar is 100 μm. d Desensitisation to auxin via the AID/JAZ degron switch rescues hPGCLC induction efficiency. Data show mean ± SD for n = 4 (no TIR1 control) or n = 11 (TIR-JAZ) biological replicates, ****P < 0.0001 (Two-way ANOVA followed by Sidak’s multiple comparison test). e Time-course of desensitisation to auxin via the AID/JAZ degron switch. Note that hPGCLC induction efficiency is only significantly rescued if Cor is supplemented from D0. Addition of Cor alone (without IAA) does not alter hPGCLC specification efficiency. Data show mean ± SD for n = 4 biological replicates, ns not significant (D0 IAA no Cor vs. D0 IAA D1 Cor P = 0.3982; D0 IAA no Cor vs. D0 IAA D2 Cor P = 0.9832), ****P < 0.0001 (Two-way ANOVA followed by Sidak’s multiple comparison test).
