Journal: bioRxiv
Article Title: The Endo-GeneScreen Platform Identifies Drug-Like Probes that Regulate Endogenous Protein Levels within Physiological Contexts
doi: 10.1101/2025.03.13.643156
Figure Lengend Snippet: A) Validation workflow encompassing luciferase-based assays, counter-screens, and protein validation assays. B-C) SR-1815 in DLR or n BIT counterscreen 8-point dose-response assay. n=12 per dose for DLR and n=10 per dose for cell free assay. Error bars represent SEM. D) Single dose Dot Blot experiment with SR-1815 treated at 1.5 µM for 14 days yielding signals for total protein and SynGAP protein expression A Mann-Whitney test was performed for total protein: U=1609, p=<1.0 e 15, and SynGAP U=0, p=<1.0 e 15. Error bars represent SEM. E-F) Dot blot dose response of SR-1815 treated for 7 days utilizing a SynGAP antibody that recognizes either all isoforms (E) or only the α2 C-terminal isoform (F); n=28 per dose. Error bars represent SEM. G) Dot Blot experiment for SynGAP protein expression in WT rat neurons. Seven-day treatment (DIV 7-14); Pan-SynGAP antibody; n=28 per dose. Error bars represent SEM. H) Western blot with samples extracted from primary cultures derived from either Syngap1 +/+ and Syngap1 +/- . Neurons were treated for 14 days with either DMSO or SR-1815 (1.5 μM); One-way ANOVA with Tukey’s post hoc tests were used. F(2,21)21.46, p=0.000008, n=8 per treatment. Error bars represent SD. I) Representative traces from mEPSC recordings in Syngap1 +/+ and Syngap1 +/- neurons treated with vehicle or SR-1815 (left) . Plot showing mEPSC frequency for the four different conditions ( middle ; 2-way ANOVA with Fisher’s LSD post hoc test, Genotype: F (1,64) = 0.2557, p = 0.6148, Treatment: F (1, 64) = 0.6138, p = 0.4362, Interaction: F (1,64) = 9.168, p = 0.0035). Plot showing mEPSC amplitudes for the four different conditions (right; 2-way ANOVA with Fisher’s LSD post hoc test, Genotype: F (1, 64) = 0.5606, p = 0.4568, Treatment: F (1, 64) = 0.5601, p = 0.4570, Interaction: F (1, 64) = 7.344, p = 0.0086). n= 17 for Syngap1 +/+ : vehicle; n= 16 for Syngap1 +/+ : SR-1815; n= 17 for Syngap1 +/- : vehicle; n= 18 for Syngap1 +/- : SR-1815. Error bars represent SEM. J) Syngap1+/+ and Syngap1+/- neurons transduced with AAV9 vectors expressing Flex-gCAMP8f and Cre (to control labeling). Neurons were treated with DMSO or 1.5 μM SR-1815 for 14 days. Calcium imaging performed on DIV14. Plot showing the spiking frequency (spikes per second) for the four different conditions (OLS regression with Tukey HSD post hoc test, Genotype: F(1,6792)=29.4, p=6.1e-8, Treatment: F(1,6792)=33.8, p=6.3e-9, Interaction: F(1,6792)=200.9, p=5.5e-45). For DMSO control 12 fields were imaged for Syngap1 +/+ Syngap1 +/- and for SR-1815 respectively 14 and 17 fields for Syngap1 +/+ Syngap1 +/- were imaged from at least 4 wells per conditions. The total number of segmented neurons were DMSO Syngap1 +/+ : 1693, Syngap1 +/- : 2047, SR-1815 Syngap1 +/+ : 1803 and Syngap1 +/- : 1253.
Article Snippet: On DIV 3 cultures were transduced with AAV9 vectors expressing pGP-AAV-syn-FLEX-jGCaMP8f-WPPRE (AAV9) (Addgene: 162379-AAV9) (MOI=300,000 vp/cell) and pENN.AAV.hSyn.Cre.WPRE.hGH (AAV9) (Addgene: 105553-AAV9) (MOI=10,000 vp/cell).
Techniques: Luciferase, Cell-Free Assay, Dot Blot, Expressing, MANN-WHITNEY, Western Blot, Derivative Assay, Transduction, Control, Labeling, Imaging
