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. 2023 Jul 27;6:0192. doi: 10.34133/research.0192

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Copyright © 2023 Cui Wang et al.

Exclusive licensee Science and Technology Review Publishing House. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY 4.0).

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Fig. 1. — Generation and characterization of MAVS-deficient pluripotent stem cells. (A) Schematic workflow showing the generation of MAVS^+/+ and MAVS^−/− hMSCs and hNSCs from hESCs. (B) Schematic illustration of MAVS gene editing in exon 2 using CRISPR/Cas9-mediated nonhomologous end joining in hESCs. (C) Western blotting analysis of MAVS protein in MAVS^+/+ and MAVS^−/− hESCs. GAPDH was used as a loading control. (D) Left: Phase-contrast images of MAVS^+/+ and MAVS^−/− hESCs. Scale bar, 250 μm. Right: Immunofluorescence staining of MAVS and TOM20 in MAVS^+/+ and MAVS^−/− hESCs. Scale bar, 25 μm. Insets show the zoomed-in images. (E) Immunofluorescence staining of pluripotency markers NANOG, SOX2, and OCT4 in MAVS^+/+ and MAVS^−/− hESCs. Scale bar, 25 μm. (F) FACS analysis of canonical markers of hMSCs, including CD73, CD90, and CD105, as well as irrelevant markers CD14, CD19, and CD34 in MAVS^+/+ and MAVS^−/− hMSCs. Cells unstained were used as the blank control. (G) Western blotting analysis of MAVS protein in MAVS^+/+ and MAVS^−/− hMSCs. GAPDH was used as a loading control. (H) Immunofluorescence staining of MAVS and TOM20 in MAVS^+/+ and MAVS^−/− hMSCs. Scale bar, 50 μm. Insets show the zoomed-in images. (I) Immunofluorescence staining of hNSC markers PAX6, SOX2, and Nestin in MAVS^+/+ and MAVS^−/− hNSCs. Scale bar, 25 μm. (J) Western blotting analysis of MAVS protein in MAVS^+/+ and MAVS^−/− hNSCs. GAPDH was used as a loading control. (K) Immunofluorescence staining of MAVS and TOM20 in MAVS^+/+ and MAVS^−/− hNSCs. Scale bar, 25 μm. Insets show the zoomed-in images. (L) Heatmap showing the Euclidean distance between replicates of RNA-seq in MAVS^+/+ and MAVS^−/−hESCs, hNSCs, and hMSCs. The color key of the Euclidean distance from blue to white indicates strong to weak correlation. (M) Principal component (PC) analysis showing the reproducibility of RNA-seq between replicates in MAVS^+/+ and MAVS^−/− hESCs, hNSCs, and hMSCs. (N) Bar plot showing the number of up-regulated (red) and down-regulated (blue) DEGs between MAVS^+/+ and MAVS^−/− hESCs, hNSCs, and hMSCs.