Figure 6. Validation of Novel hiPSC Pluripotency and Mitochondrial Factors.
(A) Pearson correlation between perturbation MDE clusters with known interactors highlighted in green for cluster 31/Pluripotency factors and cluster 20/Mitochondrial Translation. (B) Experimental workflow for metabolic tracing of mitochondrial flux validation experiment run in KOLF2.1J KRABZIM3-dCas9 transduced with sgRNAs NTC or MRPL11, or MRPL37 or ZBTB41 (C) Metabolic tracing of [U-13C]Glucose into α-Ketoglutarate isotopologues (M+0 to M+5) under different genetic perturbations. Five different single-guide RNAs (sgRNAs) were used to target MRPL11 (orange), MRPL37 (purple), ZBTB41 (yellow), and a non-targeting control (NTC, blue). The bar graph represents the percentage of isotopologue labeling from [U-13C]Glucose, showing the distribution from M+0 (unlabeled) to M+5 (fully labeled) α-Ketoglutarate. Significant differences are indicated between sgRNA-targeted genes and the NTC, as denoted by the asterisks (*p < 0.05,**p < 0.01,***p < 0.001, ****p < 0.0001). (D) Ratio of succinate to fumarate in cells transduced with various sgRNAs: MRPL11 (red), MRPL14 (green), MRPL37 (purple), ZBTB41 (yellow), and a non-targeting control (NTC, blue). Significant differences are indicated between sgRNA-targeted genes and the NTC, as denoted by the asterisks (*p < 0.05, **p < 0.01). (E) Quantitative PCR analysis of gene expression in cells transduced with sgRNAs targeting POU5F1 (purple), JOSD1 (pink), RNF7 (red), and a non-targeting control (NTC, blue). Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001). (F) KOLF2.1J KRABZIM3-dCas9 were transduced with NTC, POU5F1 or JOSD1 sgRNAs and imaged 4 days post-transduction with brightfield at 20x magnification. Top left black scale bar = 50 μm. (G) Log2 fold change (Log2FC) in gene expression from Perturb-Seq across different sgRNA treatments. The heatmap displays the Log2FC of genes (POU5F1, GRID2, CD24, DPP10, SOX5, EPHA4, GREB1L) in cells treated with sgRNAs targeting POU5F1, JOSD1, and RNF7 compared to non-targeting controls (NTCs). (H) Log2 fold change (Log2FC) in gene expression from qPCR validation across different sgRNA treatments. The heatmap shows the Log2FC of the same genes as in panel G, validated by qPCR in cells treated with sgRNAs targeting POU5F1, JOSD1, and RNF7 compared to sg-NTC. (I) Correlation between Log2FC values from Perturb-Seq and qPCR validation. The scatter plot shows the correlation of Log2FC values from Perturb-Seq and qPCR validation, with a Pearson correlation coefficient (r = 0.91) across 7 perturbations and 11 differentially expressed genes (DEGs).