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. 2023 Apr 15;14:2157. doi: 10.1038/s41467-023-37928-5

Fig. 5. Inclusion of PTBP1 exon 9 is associated with dysregulation of cellular metabolic pathways in L-HSCR-ENCCs.

Fig. 5

a Boxplot shows the percent spliced in (PSI) of exon 9 (Ex9) inclusion rate in PTBP1. Each HSCR-ENCC (HSCR#1 with 306 cells, HSCR#17 with 187 cells, and HSCR#23 with 246 cells) was compared to the merged controls with 754 cells. Each box represents the interquartile range (IQR, the range between the 25th and 75th percentile) with the mid-point of the data, whiskers indicate the upper and lower value within 1.5 times the IQR. Tests marked by “**” represent they are statistically different (two-sided Wilcoxon rank-sum test) from the controls of P values <0.01. ns not significantly different. b Scatterplot shows the enhanced and repressed exons associated with PTBP1 Ex9 enriched in L-HSCR. SE9: skipped exon 9. c Venn plots shows the PTBP1 Ex9 -enhanced and -repressed exons enriched in L-HSCR overlapping with the known PTBP1-target exons. d Gene ontology enrichment analyses of genes enriched with target exons of PTBP1-Ex9 in L-HSCR. P value and FDR were calculated by clusterProfiler software. e Schematic shows the regulation of PKM1/2 splicing regulated by PTBP1-Ex9. SE: skipped exon; MXE mutually exclusion of exon. Differentially splicing of PKM1 and PKM2 between control, S-HSCR, and L-HSCR. Two controls were merged for comparison. f RT-qPCR analyses of PTBP1, PKM1, and PKM2 expressions in ENCCs derived from the control- and various HSCR-iPSC lines. Data were presented as mean values ± SEM from 3–6 independent experiments. (Student’s t-test, two-sided). g Boxplot shows the reduced oxidative phosphorylation pathway scores in HSCR-ENCCs as inferred based on the expression of the genes implicated in these pathways. A two-sided Wilcoxon rank-sum test was applied to calculate the significance of the difference between the two groups. All samples (two controls with 754 cells, three S-HSCR with 643 cells, intermediated HSCR#1 with 306 cells, and two L-HSCR with 433 cells) were included for comparison and presented as groups. Tests marked by “****”, “***”, “**”, and “*” represent they are statistically different from the controls of P values <0.0001, <0.001, <0.01, and <0.05, respectively. ns not significantly different. Seahorse assays show oxygen consumption rate (OCR) in h control (IMR90), S-HSCR, and L-HSCR control and k control (IMR90)- and HSCR-ENCCs treated with control-, PKM1-, or PKM2- morpholinos (MO) (four independent experiments). Quantitative analyses of the i, l basal OCR and j, m maximum respiratory rate in the control and HSCR-ENCCs with or without morpholino treatment. Data were presented as mean values ± SEM from 3–10 independent experiments. (Student’s t-test, two-sided). In vitro differentiation assays show NF+ neurons derived from n control (IMR90 & UE02302)-ENCCs treated with PKM1-MO; and o HSCR-ENCCs treated with PKM2-MO, on day 5 of neuronal differentiation. Data were presented as mean values ± SEM from 3–7 independent experiments. (Student’s t-test, two-sided). Source data are provided as a Source Data file.