Fig. 1.

Establishment of TFAM-knockout hPSCs.

(A) Schematic diagram the procedure of generating Dox-induced TFAM-knockout hPSC line.

(B) Quantitative RT-PCR analysis of the expression level of TFAM and mtDNA in TFAM^-/- hPSC after Dox withdrawal. Data were shown as Mean ± SEM (n = 3, ***p < 0.001 compared with the +Dox group).

(C) Western blot of the expression of Endo-TFAM and Exo-TFAM (3 × flag tagged) protein in WT, TFAM^KI (Exo-TFAM knock-in), and TFAM^-/- hPSC with or without Dox. β-actin served as the loading control.

(D) Western blot analysis of Exo-TFAM protein level in TFAM^-/- hPSC after Dox removed. β-actin served as the loading control.

(E) Representative transmission electron photomicrographs showing the mitochondrial ultrastructure of TFAM^-/- hPSCs treated with or without Dox. Quantification of intact, impaired, and destroyed mitochondria was calculated respectively. Data were shown as Mean ± SEM (n = 15).

(F–G) Representative immunofluorescence images (F) and the quantification (G) for CellRox (green)

/ Mito-tracker (red), DAPI (blue), and JC-A (red)/M (green) in Dox treated or untreated. Scale bars, 20 μm. Data were shown as Mean ± SEM (n = 7, ***p < 0.001 compared with the +Dox group).

(H) Seahorse assay of oxygen consumption rate (OCR) in TFAM^-/- hPSCs treated with or without Dox and quantification of mitochondrial functional parameters. Data were shown as Mean ± SEM (n = 3, *p < 0.05, ***p < 0.001 compared with the +Dox group).

(I) The total ATP value in group +Dox, Short-Term, and Long-Term. Data were shown as Mean ± SEM (n = 3, ***p < 0.001 compared with the +Dox group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)