Fig. 4
Optimization and application of HiHo-AID2 in hESCs. a Scheme illustrating the two promoters P1 and P2 in plasmids for one-step generation of AID cells. b Graphs showing the cell count, fraction of mCherry-positive and GFP-positive cells using different P1 and P2 promoters in hESCs. N = 3 technical repeats. c, d Graphs showing the percentage of GFP-positive cells, the percentual change of single-cell GFP intensity compared to control (coIN) and the cell count (× 105 cells/ml) with different HDR enhancers with or without P53DD. P53DD: a dominant-negative P53 mutant; numbers above columns indicate mean values; lines link the same endogenous tagging pairs. N.A.: not available due to insufficient number of cells for FACS analysis. N = 5 (c) and 6 (d) different protein targets. All statistical comparisons are shown in Additional file 4: Table S6. e Graph depicting the genotyping PCR results for AID clones generated with HiHo-AID2 in hESCs using i53 plus 0.25 μM M3814 as HDR enhancers in the absence of P53DD. Clones were generated as indicated in Fig. 2f, g. Number above each column indicates total amount of clones analyzed. f–j WB analysis of inducible degradation of 5 target proteins in degron hESCs. k Graph showing the PI4P staining intensity of wild-type and SAC1 degron hESCs. N = 13, 9, 18, 20 fields respectively. One-way ANOVA, n.s.: non-significant, **** p < 0.001. All statistical comparisons are shown in Additional file 4: Table S6. l Lipid droplet (LD) staining with LD540 in wild-type and BSCL2 degron hESCs. Oleic acid (0.2 mM) was added during the final 4 h to induce LD formation. Scale bar: 5 μM. m Live-cell imaging analysis of morphological changes and cell death in wild-type and 3 different degron hESCs. Representative images in different time points from the same areas are shown. Scale bar: 50 μM; N = 4 fields. n WB analysis with anti-PMP70 and anti-miniIAA7 antibodies in wild-type and PEX3 degron hESCs. Arrow indicates the specific PMP70 protein bands. Representative of 2 clones for each target protein (f–n). WT: wild-type; pico: 0.5 μM pico_cvxIAA treatment; a.u. arbitrary unit