Fig. 1

Generation of TXNIP−/− and TXNIP+/+ H1-hESCs. A Schematic representation of the CRISPR-Cas12a-mediated gene editing strategy targeting exon 3 of the TXNIP gene in H1-hESCs along with the PCR primer binding regions. B PCR analysis using internal and external primers showing the successful deletion of exon 3 in the TXNIP+/−, and TXNIP−/− clones, with TXNIP+/+ as the wild-type control. C Confirmation of the deleted exon 3 region by Sanger sequencing in selected TXNIP−/− and TXNIP+/+ clones from the PCR analysis. D qPCR showing absence of TXNIP mRNA expression in TXNIP−/− H1-hESCs (n = 4, mean ± SEM, **p < 0.01, unpaired student’s t-test). E Representative western blot and quantification confirming the absence of TXNIP protein expression in TXNIP−/− H1-hESCs (n = 3, mean ± SEM, *< 0.05, unpaired student’s t-test). Full-length blots are presented in Supplementary Figure S5A. F Representative immunofluorescence images showing comparable expression of pluripotency markers (OCT4, NANOG, TRA-1–60, and SSEA-4) in TXNIP−/− and TXNIP+/+ H1-hESCs. Scale bar, 50 μm. G Karyotyping analysis indicates chromosomal integrity is maintained in TXNIP−/− and TXNIP+/+ H1-hESCs. H Representative immunofluorescence images showing successful differentiation of TXNIP−/− and TXNIP+/+ hESCs into endoderm (SOX17), mesoderm (VIM), and ectoderm (TUBB3). Scale bar, 50 μm