![Residual haplotype-specific MAPK3 expression, transcriptomics profile and electrophysiological properties in 16p11.2del cortical NCs. (A) Quantification of ERK1/pERK1/Kip1(p27) protein levels in 16p11.2del NPCs carrying different residual haplotypes. Level was normalized by β-actin. Del_H1: n = 4 clones. Del_H2: n = 4 clones. (B) Quantification of MAPK3 mRNA levels in 16p11.2del NPCs carrying different residual haplotypes before and after treatment with MAPK3 lentiviral expression vectors. NPCs were collected for RNA extraction, genomic DNA digestion following RT-qPCR. MAPK3 mRNA expressions were normalized by GAPDH. Del_H1: n = 4 clones, Del_H2: n = 4 clones, Del_H1_R (+): n = 2 clones, Del_H2_R (+): n = 2 clones. (C–E) Quantification analysis of ERK1 (C), pERK1 (D), Kip1(p27) (E) protein levels in 16p11.2del NPCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (+): n = 2 clones, Del_H1_R (++): n = 2 clones, Del_H2_R (+): n = 2 clones, Del_H2_R (++): n = 2 clones] or empty (Del_H1: n = 2 clones, Del_H2: n = 2 clones) lentiviral expression vectors. 16p11.2del cells treated with EGFP expression vector (4 × 105 TU) were used for lentiviral control. Two dosages of MAPK3 expression vectors were performed. R (+): 8 × 104 TU, R (++): 4 × 105 TU. The comparisons were performed for different subgroups. All values were normalized by β-actin. (F) Cumulative differences of weighted mean firing rates of 16p11.2del NCs carrying different residual haplotypes. Electrophysiological signatures were recorded by MEA. The x-axis shows timeline after plating NPCs (Days 9 to 33), Del_H1: n = 2 clones. Del_H2: n = 3 clones. The cumulative differences or the difference at each observation time of weighted mean firing rates of NCs (Days 9 to 33) was compared by ANOVA of Aligned Rank Transformed Data. (G and H) Representative images of soma size in 16p11.2del NCs carrying different residual haplotypes (G) and quantification analysis (H). Del_H1: n = 68 cells. Del_H2: n = 50 cells. ***P < 0.001, **P < 0.01, *P < 0.05. (I and J) Representative images and quantification analysis of soma size in 16p11.2del NCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (+): n = 113 cells, Del_H1_R (++): n = 131 cells, Del_H2_R (+): n = 134 cells, Del_H2_R (++): n = 80 cells] or empty (Del_H1: n = 85 cells, Del_H2: n = 94 cells) lentiviral expression vectors. 16p11.2del cells treated with EGFP expression vector (4 × 105 TU) were used for lentiviral control. Two dosages of MAPK3 expression vectors were performed. R (+): 8 × 104 TU, R (++): 4 × 105 TU. ** P < 0.01. (K and L) Quantification of weighted mean firing rates in 16p11.2del NCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (++): n = 2 clones, Del_H2_R (++): n = 3 clones] or control (Del_H1: n = 2 clones, Del_H2: n = 3 clones) lentiviral expression vectors. Each cell clone was treated only using R (++) dosage (4 × 105 TU). The x-axis shows timeline after seeding NPCs. The cumulative differences or the difference at each observing time of weighted mean firing rates of NCs (Days 9 to 33) was compared by ANOVA of Aligned Rank Transformed Data. 16p11.2del cells treated with EGFP empty expression vector (4 × 105 TU) were used for control. ***P < 0.001, **P < 0.01, *P < 0.05. Each box and whisker plot indicates median (line within box), mean (+), interquartile range (box outline), maximum and minimum values (whiskers).](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/brain/146/8/10.1093_brain_awad071/1/awad071f5.jpeg?Expires=1750830125&Signature=n6F0o4xpK0uxJzM30vQnbq678pwYVT5LU65Zxdv27PFnZhAssQhD-4yracO0tb0tV244AsmvHXwTAzmV4gcqqKFsiFBIq6WEFa7S~DskxJVEb2O-Adyfa1mI3gDkX7aCr6y93tFePp~7N1tuWqSz4P2VFmJSANt2mgxw5Qhu6UiZDWLRVNo0vKF3Gq0ePuBNxeLBewU180IDhlfXDHBuXn-~Ezj0CEYGyN8zleSsZd2k5ClDBQICjnyuMwkgqP-CZztvKdSh2grtdFOLEcMCuBcFWN4zdeOlNNPCEECsuMwr54LiayKOiFmGMSixtnYHrekMvL1nO~9nwwizXoo8~A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Residual haplotype-specific MAPK3 expression, transcriptomics profile and electrophysiological properties in 16p11.2del cortical NCs. (A) Quantification of ERK1/pERK1/Kip1(p27) protein levels in 16p11.2del NPCs carrying different residual haplotypes. Level was normalized by β-actin. Del_H1: n = 4 clones. Del_H2: n = 4 clones. (B) Quantification of MAPK3 mRNA levels in 16p11.2del NPCs carrying different residual haplotypes before and after treatment with MAPK3 lentiviral expression vectors. NPCs were collected for RNA extraction, genomic DNA digestion following RT-qPCR. MAPK3 mRNA expressions were normalized by GAPDH. Del_H1: n = 4 clones, Del_H2: n = 4 clones, Del_H1_R (+): n = 2 clones, Del_H2_R (+): n = 2 clones. (C–E) Quantification analysis of ERK1 (C), pERK1 (D), Kip1(p27) (E) protein levels in 16p11.2del NPCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (+): n = 2 clones, Del_H1_R (++): n = 2 clones, Del_H2_R (+): n = 2 clones, Del_H2_R (++): n = 2 clones] or empty (Del_H1: n = 2 clones, Del_H2: n = 2 clones) lentiviral expression vectors. 16p11.2del cells treated with EGFP expression vector (4 × 105 TU) were used for lentiviral control. Two dosages of MAPK3 expression vectors were performed. R (+): 8 × 104 TU, R (++): 4 × 105 TU. The comparisons were performed for different subgroups. All values were normalized by β-actin. (F) Cumulative differences of weighted mean firing rates of 16p11.2del NCs carrying different residual haplotypes. Electrophysiological signatures were recorded by MEA. The x-axis shows timeline after plating NPCs (Days 9 to 33), Del_H1: n = 2 clones. Del_H2: n = 3 clones. The cumulative differences or the difference at each observation time of weighted mean firing rates of NCs (Days 9 to 33) was compared by ANOVA of Aligned Rank Transformed Data. (G and H) Representative images of soma size in 16p11.2del NCs carrying different residual haplotypes (G) and quantification analysis (H). Del_H1: n = 68 cells. Del_H2: n = 50 cells. ***P < 0.001, **P < 0.01, *P < 0.05. (I and J) Representative images and quantification analysis of soma size in 16p11.2del NCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (+): n = 113 cells, Del_H1_R (++): n = 131 cells, Del_H2_R (+): n = 134 cells, Del_H2_R (++): n = 80 cells] or empty (Del_H1: n = 85 cells, Del_H2: n = 94 cells) lentiviral expression vectors. 16p11.2del cells treated with EGFP expression vector (4 × 105 TU) were used for lentiviral control. Two dosages of MAPK3 expression vectors were performed. R (+): 8 × 104 TU, R (++): 4 × 105 TU. ** P < 0.01. (K and L) Quantification of weighted mean firing rates in 16p11.2del NCs carrying different residual haplotypes after treatment with MAPK3 [Del_H1_R (++): n = 2 clones, Del_H2_R (++): n = 3 clones] or control (Del_H1: n = 2 clones, Del_H2: n = 3 clones) lentiviral expression vectors. Each cell clone was treated only using R (++) dosage (4 × 105 TU). The x-axis shows timeline after seeding NPCs. The cumulative differences or the difference at each observing time of weighted mean firing rates of NCs (Days 9 to 33) was compared by ANOVA of Aligned Rank Transformed Data. 16p11.2del cells treated with EGFP empty expression vector (4 × 105 TU) were used for control. ***P < 0.001, **P < 0.01, *P < 0.05. Each box and whisker plot indicates median (line within box), mean (+), interquartile range (box outline), maximum and minimum values (whiskers).
