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. Author manuscript; available in PMC: 2024 Apr 2.
Published in final edited form as: Cell Rep. 2022 May 3;39(5):110790. doi: 10.1016/j.celrep.2022.110790

Figure 5. SETD1A haploinsufficiency leads to activation of the cAMP/PKA/CREB pathway.

Figure 5

(A) GO terms related to second messenger signaling associated with upregulated DEGs.

(B) Change of mRNA levels of genes coding for adenylyl cyclase (AC) and phosphodiesterases.

(C) Schematic of the cAMP/PKA/CREB pathway and drug targets at different steps.

(D) cAMP concentrations for control (n = 6), control + 1 μM FSK (n = 7), and SETD1A+/− cultures (n = 7 from clone 1).

(E) Western blot of pCREB, CREB, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for control (n = 8), control + 1 μM FSK (n = 4), and SETD1A+/− cultures (n = 8 from clone 1).

(F and G) Representative images showing pCREB expression and quantification of intensity of pCREB for control (n = 134 cells), control + 1 μM FSK (n = 96 cells),and SETD1A+/− neurons (n = 156 cells, from 3 independent batches; n = 34 from clone 1 and n = 122 from clone 2). Scale bar, 10 μm.

(H and I) Representative raster plot for 30 s recorded by MEA and quantification of network burst rate, showing the effect of the AC agonist FSK (1 μM) on control networks (sample size: control n = 10 wells, control + FSK n = 12 wells, SETD1A+/− n = 28 wells [n = 6 from clone 1 and n = 22 from clone 2]).

(J and K) Representative raster plot for 30 s recorded by MEA and quantification of network burst rate, showing the effect of the AC inhibitor SQ22536 (100 μM) on SETD1A+/− E/I cultures (sample size: control n = 11 wells, SETD1A+/− + SQ22536 n = 13 wells, SETD1A+/− n = 13 wells from clone 2).

(L and M) Representative raster plot for 30 s recorded by MEA and quantification of network burst rate, showing the effect of the PKA inhibitors H89 (2 μM) and KT5720 (1 μM) on SETD1A+/− E/I cultures (sample size: control n = 15 wells, SETD1A+/− + H89 n = 18 wells [n = 3 from clone 1 and n = 15 from clone 2], SETD1A+/− + KT5820 n = 8 wells [n = 2 from clone 1 and n = 6 from clone 2], SETD1A+/− n = 22 wells [n = 7 from clone 1 and n = 15 from clone 2]).

Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test and post hoc Bonferroni correction.